Compositions and methods for protecting animals from lentivirus-associated disease such as feline immunodeficiency virus

ABSTRACT

The present invention is directed to a novel strain of feline immunodeficiency virus, designated herein as FIV-141, and to attenuated forms of the virus produced by mutating specific regions of the viral genome. The virus and mutated forms of the virus may be used to induce the production of antibodies to FIV-141, and in vaccines designed to protect cats from FIV.

This application claims priority from U.S. provisional applicationSerial No. 60/097,645, filed Aug. 24, 1998 under 35 U.S.C. §119(e).

FIELD OF THE INVENTION

The present invention is directed to a novel strain of felineimmunodeficiency virus (FIV) and to a variety of mutated forms of thisvirus. Compositions and methods are disclosed that can be used in theprotection of animals from lentiviral associated disease.

BACKGROUND OF THE INVENTION

Feline immunodeficiency virus (FIV) infection in cats results in adisease syndrome that is similar to that caused by humanimmunodeficiency virus-1 (HIV-1) infection in humans. Diseaseprogression begins with a transient acute phase (8-10 weeks), followedby a prolonged asymptomatic phase (lasting from weeks to years) and aterminal symptomatic phase (Ishida et al., 1990, Jpn. J. Vet. Sci.52:645-648). Viral load in plasma has been demonstrated to correlatewith disease stage in infected cats and can be used to predict diseaseprogression in accelerated FIV infection (Diehl et al., 1996, J. Virol.70:2503-2507).

Structurally, the FIV provirus contains two long terminal repeats(LTRs), one at either end of the genome (Talbott et al., 1989, Proc.Nat'l Acad. Sci. USA 86:5743-5747). There are three large open readingframes (Gag (group antigens); Pol (polymerase); and ENV (envelope)) andthree small open reading frames encoding regulatory proteins (Rev(regulator of expression of virion, a protein that binds to “RRE”elements present in all viral transcripts and promotes theirtranslocation from the nucleus to the cytoplasm of infected host cells);Vif (virion infectivity factor); and ORF(2) (open reading frame 2)). TheGag precursor polypeptide of FIV is processed into three maturestructural proteins: a matrix protein (MA), a capsid protein (CA), and anucleocapsid protein (NC). The Pol gene encodes four enzymatic proteins:a protease (PR), a reverse transcriptase (RT), a deoxyuridinetriphosphatase (DU), and an integrase (IN). Finally, the ENV precursorpolypeptide is processed into two envelope proteins: a surface protein(SU) and a transmembrane (TM) protein.

There have been several attempts to develop a safe and effective vaccineto FIV. Matteucci found that cats inoculated with a conventional fixedcell vaccine were protected from challenge with homologous virus despitean apparent absence of neutralizing antibodies after vaccination.Protection was found to be short-lived and difficult to boost (Matteucciet al., 1996, J. Virol. 70:617-622; Matteucci et al., 1997, J. Virol.71:8368-8376). These results may be contrasted with those of Verschoor,who observed no protection after the administration of a fixed cellvaccine (Verschoor et al., 1995, Vet. Immunol. Immunopathol.46:139-149).

Another type of conventional vaccine that has been tested is comprisedof whole, inactivated FIV virus. Yamamoto reported that greater than 90%of cats administered a vaccine of this type exhibited essentiallycomplete protection against homologous challenge and slight protectionagainst heterologous virus (Yamamoto et al., 1993, J. Virol.67:601-605). Both humoral and cellular immunity against FIV were inducedand a high level of anti-ENV, anti-core and virus neutralizing (VN)antibodies were observed in the vaccinated cats. In contrast,vaccination of cats with inactivated whole FIV incorporated into immunestimulating complexes (ISCOMs) failed to protect animals from homologouschallenge (Hosie et al., 1992, Vet. Immunol. Immunopathol. 35:191-197).

Another approach to vaccine development has involved the use of subunitvaccines containing recombinant core protein, synthetic V3 peptides, andrecombinant ENV protein (Elyar et al., 1997, Vaccine 15:1437-1444).Although significant levels of antibodies were induced by such vaccines,none were identified that could protect cats against homologous FIVchallenge (Huisman et al., 1998, Vaccine 16:181-187; Flynn et al., 1997,J. Virol. 71:7586-7592; Tijhaar et al., 1997, Vaccine 15:587-596). Theresults suggest that it is likely to be difficult to obtain protectiveimmunity against FIV using subunit type vaccines.

Recently, Cuisinier reported on tests conducted on a DNA vaccine for FIV(Cuisinier et al., 1997, Vaccine 15:1085-1094). Cats were vaccinatedwith a plasmid carrying FIV structural genes, including ENV and p10.Although strong humoral immune responses were observed, all catseventually succumbed to homologous challenge.

SUMMARY OF THE INVENTION

The present invention is based, in part, upon the isolation andcharacterization of a new strain of feline immunodeficiency virus,designated herein as FIV-141 and deposited as ATCC No. VR-2619. Thecomplete genomic sequence of the virus has been determined and isdistinct from all other known FIV sequences. A plasmid encoding FIV-141has been deposited as ATCC No. 203001.

A. Compositions and Methods Based Upon the FIV-141 Virus

In its first aspect, the present invention is directed to asubstantially purified FIV-141 virus having a genomic sequencecorresponding to that of SEQ ID NO:1, to host cells infected with thevirus and to progeny virus produced in the host cells. The term“substantially purified” means that FIV-141 has been separated from allother strains of virus and, particularly, from all other strains of FIV.Host cells are typically cells grown in in vitro culture. Host cellsthat may be used for growing virus include peripheral blood mononuclearcells (PBMCs). Progeny virus may be isolated using standard proceduresas discussed below. The present invention further provides asubstantially purified virus having a nucleotide sequence which is adegenerate variant of a nucleotide sequence corresponding to SEQ IDNO:1, as based on the degeneracy of the genetic code, host cellsinfected with such a virus, and progeny virus produced in the hostcells, which are useful for all of the purposes disclosed herein for thesubstantially purified FIV-141 virus having a genomic sequencecorresponding to that of SEQ ID NO:1, and for which all of thedisclosure provided herein below is equally applicable.

The FIV-141 virus and host cells infected with the virus can be used toinfect animals for the purpose of inducing the production of antibodiesthat react preferentially with one or more strains of FIV. “Preferentialbinding” of antibodies, as used herein, refers to an antibody having atleast a 100-fold greater affinity for FIV than for any other virus ornon-FIV protein. Antibodies may be generated in any of the animalscommonly used for this purpose (such as, e.g., mice, rabbits, goats, orsheep) but, preferably, antibodies will be made in domestic cats. Whenvirus is used to induce antibody production, it may, if desired, beinactivated prior to infection. Inactivation procedures may involvetreating the virus with formalin, paraformaldehyde, phenol,lactopropionate, ultraviolet light, heat, psorlens, platinum complexes,ozone or other viricidal agents. When host cells expressing FIV-141 areused to induce antibody production, the cells may be fixed prior toinfection. Typically, this will involve treating the cells withparaformaldehyde as described herein, but other methods may also beemployed. Antibodies made to FIV-141 are themselves included within thescope of the invention and may be purified using techniques well knownin the art (see, e.g., Harlow et al., 1988, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, N.Y.).

In another aspect, the invention is directed to a whole virus vaccinecomprising inactivated FIV-141 virus, or an inactivated virus encoded bya degenerate variant of a nucleic acid molecule having a nucleotidesequence corresponding to SEQ ID NO:1. An immune response may be inducedin a cat by administering this vaccine at a dosage and for a durationsufficient to induce protective immunity against subsequent infectionwith FIV-141. Typically, the vaccine will be administered parenterallywith two or more inoculations being given at intervals of, e.g., two toeight weeks. The invention also includes a fixed cell vaccine, which iscomprised of a host cell infected with the FIV-141 virus or a degeneratevariant thereof. Administration of this vaccine will follow the samegeneral procedures as used for the whole virus vaccine. Standardprocedures well known in the art may be used to optimize immunizationprotocols.

B. Compositions and Methods Based Upon FIV-141 Genomic Nucleic Acid

In another aspect, the present invention is directed to a substantiallypurified nucleic acid molecule (DNA or RNA) having a sequencecorresponding to that of SEQ ID NO:1 or a degenerate variant thereof. Asused in this context, “substantially purified” means that the desiredproduct is essentially free from contaminating cellular components. A“substantially pure” nucleic acid will typically comprise at least 85%of a sample, with greater percentages being preferred. Contaminants mayinclude proteins, carbohydrates or lipids. One method for determiningthe purity of a nucleic acid is by electrophoresing a preparation in amatrix such as polyacrylamide or agarose. Purity is evidenced by theappearance of a single band after staining. Other methods for assessingpurity include chromatography and analytical centrifugation. The FIV-141nucleic acid may be used in place of the whole virus to transfect hostcells and to thereby induce the production of progeny virus or viralproteins.

The invention also encompasses methods of inducing the production ofantibodies to FIV-141 by injecting nucleic acid directly into an animalor by administering host cells transfected with the nucleic acid. Aswith the procedures discussed above in connection with the whole virus,host cells may be fixed prior to administration. Antibodies may besubstantially purified from animals and used in assays designed todetect the presence of FIV in culture medium or in a biological fluid. A“substantially purified” antibody will typically comprise at least 70%of protein in a sample, with greater percentages being preferred.

Host cells transfected with FIV-141 genomic DNA or a degenerate variantthereof may also be used in a vaccine for immunizing cats. If desired,such cells may be fixed to reduce viral infectivity, e.g., by treatmentwith an agent such as paraformaldehyde. Vaccines made in this manner maybe used to induce an immune response in a cat. The vaccine may beadministered using a standard immunization protocol optimized for theinduction of protective immunity against subsequent infection withFIV-141 or, if desired, some other strain of FIV.

C. Attenuated FIV-141 Virus and Vaccines

Before a whole virus can be administered to an animal as a vaccine, itmust be converted into a non-pathogenic form. As discussed above, thismay be accomplished by inactivating the virus or fixing host cells. Analternative method involves introducing mutations into the virus totransform it into an attenuated form. The phrase “attenuated virus” asused in this context, refers to a virus that has substantially reducedinfectivity compared to its wild type counterpart. Infectivity may bemeasured in PBMCs, as described in the Examples section herein below.

Thus, the invention is directed to an attenuated FIV-141 virus, ordegenerate variant thereof, that exhibits significantly reducedinfectivity for feline T lymphocytes relative to the wild type (i.e.,non-mutated) virus. The attenuated virus is produced by mutating one ormore genes in the FIV-141 genome or degenerate variant thereof selectedfrom the group consisting of Vif, MA, ORF(2), ENV, CA, NC, SU, TMf, CT,IN, DU, V3/4, V7/8 and RRE. Appropriate mutations for each of thesegenes are described herein. Examples of several specific mutations thatmay be used in making attenuated viruses include MA del, ENV del, V3/4del, V7/8 del, TMF del, CT del, Vif del, Vifc del, Vifn del, ORF(2) del,CA del, NC del, IN del, DU del, SU del, and RRE del. In a preferredembodiment, the attenuated virus comprises a mutation in the ENV gene.In a further preferred embodiment, the attenuated virus comprises acombination of any two or more of the aforementioned mutations. Inspecific though non-limiting embodiments, the attenuated virus comprisesdouble mutations in any of the following combinations of genes: (i)MA/TMf; (ii) MA/V3/4; (iii) MA/Vif; or (iv) ENV/IN. In a preferredembodiment, the attenuated virus comprises any of the following doubledeletions: (i) MA del/TMf del; (ii) MA de/V3/4 del; (iii) MA de/Vif delor (iv) ENV del/IN del. In a further preferred embodiment, theattenuated virus comprises at least two mutations, one of which is inthe ENV gene such as, e.g., ENV del, with one or more other mutations inany of the other genes of the virus. In a preferred embodiment, the oneor more other mutations in the other genes of the virus are in genesselected from the group consisting of IN, CA, NC, Vif and ORF(2).

The invention also encompasses host cells infected with the attenuatedvirus and the progeny virus produced by such cells. Once produced, theattenuated virus may be purified from host cells using standardprocedures.

Antibody production may be induced by infecting an animal with theattenuated virus or, alternatively, infected host cells may be used. Ifdesired, the virus may be inactivated or the host cells fixed prior toadministration to an animal and antibodies may be purified from animalsusing standard procedures.

In addition, the invention encompasses a vaccine that utilizes theattenuated whole virus discussed above or a host cell infected with oneof these viruses. Again, the attenuated viruses may be inactivated andthe host cells may be fixed. Such treatments may provide added assurancethat vaccines will not themselves cause infection. Vaccines based uponone or more attenuated FIV-141 viruses or degenerate variants thereofmay be used to induce protective immunity in a cat. Standardimmunization protocols may be followed in administering vaccines so asto optimize the induction of protective immunity against subsequentchallenge with FIV-141.

D. Compositions and Methods Based Upon Mutated FIV-141 Genomic DNA

In another aspect, the present invention is directed to a substantiallypurified FIV-141 nucleic acid (DNA or RNA) having a sequencecorresponding to SEQ ID NO:1, or degenerate variant thereof, but whichhas been mutated to encode an attenuated virus. Mutations should be toone or more genes selected from the group consisting of Vif, MA, CA, NC,SU, TMf, ORF(2), CT, ENV, Vifn, Vifc, IN, DU, V3/4, V7/8, and RRE, andshould be made in such a manner that, upon introduction into a hostcell, a virus is made that has significantly reduced infectivity forfeline T lymphocytes (or other susceptible cell types) relative to thewild type virus. Examples of several specific mutations that willproduce an appropriate attenuated virus are described herein andinclude: MA del, ENV del, V3/4 del, V7/8 del, TMf del, CT del, Vif del,Vifc del, Vifn del, ORF(2) del, SU del, CA del, NC del, IN del, DU del,and RRE del. In a preferred embodiment, the attenuated virus comprises acombination of any two or more of the aforementioned mutations. Inspecific though non-limiting embodiments, the attenuated virus comprisesdouble mutations in any of the following combinations of genes: (i)MA/TMf; (ii) MA/V3/4; (iii) MA/Vif; or (iv) ENV/IN. In a preferredembodiment, the attenuated virus comprises any of the following doubledeletions: (i) MA del/TMf del; (ii) MA del/V3/4 del; (iii) MA de/Vif delor (iv) ENV del/IN del. In a further preferred embodiment, theattenuated virus comprises at least two mutations, one of which is inthe ENV gene such as, e.g., ENV del, with one or more other mutations inany of the other genes of the virus. In a preferred embodiment, the oneor more other mutations in the other genes of the virus are in genesselected from the group consisting of IN, CA, NC, Vif and ORF(2).

The invention includes host cells transfected with the mutated nucleicacid and FIV progeny virus produced by the host cells.

Also included within the scope of the invention is a method of inducingthe production of antibodies to FIV in an animal by injecting eithernucleic acid mutated as described above or a host cell that has beentransfected with the nucleic acid. If desired, the host cell may befixed prior to administration. The antibodies produced may be purifiedusing standard methods and used in assays designed to detect thepresence of FIV.

The nucleic acid, preferably DNA, which has been mutated, may be used ina vaccine in which it is present at a concentration sufficient to induceprotective immunity upon administration to a cat. Alternatively, avaccine may include a host cell transfected with such DNA and, ifdesired, the host cell may be fixed after viral proteins are expressed.The vaccines may be administered to a cat at a dosage and for a durationsufficient to induce an immune response in a cat, and immunizationprotocols may be optimized for inducing protective immunity againstsubsequent infection by FIV-141.

E. Methods of Making and Using Attenuated Lentiviruses

The methods disclosed herein in connection with the production ofattenuated FIV-141 may be effectively applied to the attenuation ofother strains of FIV and to other types of lentivirus. A virus that hassignificantly reduced infectivity relative to its unmutated, wild typecounterpart may be made by mutating one or more genes selected from thegroup consisting of MA, CA, NC, DU, ENV, SU, TMf, CT, Vif, ORF(2), Vifn,Vifc, IN, V3/4, V7/8, and RRE. In a preferred embodiment, the attenuatedlentivirus comprises a mutation in the ENV gene. In a further preferredembodiment, the attenuated virus comprises a combination of any two ormore of the aforementioned mutations. In specific though non-limitingembodiments, the attenuated virus comprises double mutations in any ofthe following combinations of genes: (i) MA/TMf; (ii) MA/V3/4; (iii)MA/Vif; or (iv) ENV/IN. In a preferred embodiment, the attenuated viruscomprises any of the following double deletions: (i) MA del/TMf del;(ii) MA del/V3/4 del; (iii) MA de/Vif del or (iv) ENV del/IN del. In afurther preferred embodiment, the attenuated virus comprises at leasttwo mutations, one of which is in the ENV gene such as, e.g., ENV del,with one or more other mutations in any of the other genes of the virus.In a preferred embodiment, the one or more other mutations in the othergenes of the virus are in genes selected from the group consisting ofIN, CA, NC, Vif and ORF(2).

Mutations should be designed to eliminate or substantially reduce theactivity of the gene product. This may be accomplished by deleting theentire gene or by deleting a large (e.g., one fourth) fraction of theentire gene. The invention encompasses the attenuated lentiviruses madeusing the disclosed methods, host cells infected with these viruses, andmethods of inducing antibody production by injecting the attenuatedvirus into a mammal. The antibodies may be purified from infectedmammals and used in immunoassays. Alternatively, the purifiedantibodies, or antibody-containing serum derived from animals infectedwith attenuated virus, may be used to treat a mammal for lentivirusinfection.

As used herein, the phrase “induction of protective immunity”, and thelike, is used broadly to include the induction of any immune-basedresponse in response to vaccination, including either an antibody orcell-mediated immune response, or both, that serves to protect thevaccinated mammal against the particular lentivirus. The terms“protective immunity”, “protective response”, “protect”, and the like,as used herein, refer not only to the absolute prevention of any of thesymptoms or conditions in the mammal resulting from infection with theparticular lentivirus, but also to any detectable delay in the onset ofany such symptoms or conditions, any detectable reduction in the degreeor rate of infection by the particular lentivirus, or any detectablereduction in the severity of the disease or any symptom or conditionresulting from infection by the particular lentivirus. Vaccinepreparations according to the present invention should be administeredat a dosage and for a duration sufficient to reduce one or more clinicalsigns and viral load associated with the infection of the mammal. Whenthe lentivirus is a strain of FIV, the mammal treated will be a cat andthe signs associated with the infection will include immunologicalabnormalities such as an abnormally low level of CD4⁺ T-lymphocytes oran abnormally elevated number of CD8⁺ T-lymphocytes. Other clinicalsigns will typically include alopecia, anemia, chronic rhinitis,conjunctivitis, diarrhea, emaciation, enteritis, gingivitis,hematochezia, neurological abnormalities and dermatitis.

The attenuated lentivirus (e.g., an attenuated strain of FIV), or hostcells infected with such a virus, may be used in vaccine at aconcentration sufficient to induce immunity when administered to amammal (e.g., a cat). An immune response may then be induced byadministering such a vaccine at a dosage and for a duration sufficientto induce protective immunity against subsequent infection by at leastone strain of lentivirus.

F. Methods of Making and Using Mutated Lentivirus Nucleic Acid

The present invention is also directed to a method of producing anucleic acid suitable for use in a vaccine against lentivirus, e.g. FIV,infection. This is accomplished by reverse transcribing the genomic RNAof the lentivirus; cloning the reverse transcript; mutating one or moregenes selected from the group consisting of MA, CA, NC, SU, TMf, ORF(2),CT, ENV, Vif, Vifn, Vifc, V3/4, V7/8, IN, DU, and RRE; and then cloningthe mutated nucleic acid. Preferably, mutations should be such that,upon introduction into a host cell, an attenuated virus is made that hassignificantly reduced infectivity relative to lentivirus produced by theunmutated, wild type nucleic acid. In the case of FIV, infectivityshould be reduced or eliminated for feline T-lymphocytes.

Mutated lentivirus nucleic acid may be purified and used to transfecthost cells, make progeny virus, and make antibody in the same way asdescribed above for FIV-141. In addition, the nucleic acid, or hostcells transfected with the nucleic acid, may be incorporated into avaccine and used to induce protective immunity in a mammal. Preferably,the nucleic acid will encode an attenuated strain of FIV that hassignificantly reduced infectivity in feline PBMCs, includingT-lymphocytes such as FeP2 cells. Under these circumstances, the immuneresponse will be induced in a cat.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: FIV Production from Transfected Cells. Crandell Feline Kidney(CRFK) cells were transfected with a plasmid comprising the full lengthFIV-141 genome. Beginning at 24 hours post-transfection, cellsupernatants were harvested and assayed for the presence of FIV p26capsid protein using an enzyme immunoassay.

FIG. 2: Infection of FeP2 T Lymphocytes by Co-culture. CRFK cells weregrown in six well plates and transfected with plasmid DNA encoding thefull length FIV-141 genome. Forty-eight hours after transfection, 2×10⁶FeP2 cells were introduced into each well. Beginning 72 hours afterco-cultivation, FeP2 cells were separated and their supernatants weretested for the presence of FIV p26 capsid protein by ELISA.

FIG. 3: Infection of FeP2 Cells by Adsorption. 2×10⁶ FeP2 cells weresuspended in 200 ul of FIV-141 virus-containing conditioned mediumderived from CRFK cells transfected with the full length infectiousFIV-141 clone. Beginning at four days post-infection, FeP2 cellsupernatants were tested for the presence of FIV-141 virus using a p26ELISA assay.

FIG. 4: Expression of FIV-141 Viral Protein by Deletion Clones. Avariety of clones mutated to delete FIV genes or regulatory regions weremade and transfected into CRFK cells. After 48 hours, cell supernatantswere assayed for the presence of FIV p26 capsid protein by ELISA. Theresults for each deletion clone, as well as the wild type FIV-141molecular clone, are shown.

FIGS. 5-10: Infection of FeP2 T Lymphocytes by FIV-141 Mutants. CRFKcells were grown in six well plates and infected with one of threedifferent FIV-141 deletion clones: TMf del, ENV del, or NC del. After 48hours, FeP2 cells were added to each well and co-cultures weremaintained for an additional 72 hours. The FeP2 cells were thenseparated from the CRFK cells and assayed for the presence of p26antigen using an ELISA assay. Monitoring of p26 levels was repeatedevery 3-4 days and results are shown in FIG. 5. The experiment wasrepeated using: Vifn del, Vifc del and Vif del (FIG. 6); MA del and CAdel (FIG. 7); V3/4 del, V7/8 del and CT del (FIG. 8); ORF(2) del (FIG.9); and DU del, SU del, IN del, and RRE del (FIG. 10).

FIG. 11: Cumulative viral RNA loads detected by QcRT-PCR in plasmasamples obtained from cats vaccinated with plasmids encoding particulardeletion mutants of FIV-141 and then challenged with FIV-141. Cats inthe positive placebo group were vaccinated with pCR-Script SK(+) vectorDNA and then challenged with FIV-141. Cats in the negative placebo groupwere vaccinated with pCR-Script SK(+) vector DNA, but were notchallenged with FIV-141.

DETAILED DESCRIPTION OF THE INVENTION A. Production of FIV-141 and DNAEncoding the Virus

The present invention is directed to a novel strain of felineimmunodeficiency virus (designated herein “FIV-141”) that isdistinguished from all similar strains based upon its genomic nucleicacid sequence and biological functions. Although the genome of FIV-141consists of RNA, this is reverse transcribed into DNA and integratedinto the genome of an infected host. It will be understood thatreferences made herein to sequences of FIV and to mutated forms of suchsequences encompass both the reverse transcribed viral RNA sequences aswell as the corresponding DNA itself.

It is well known that techniques such as site-directed mutagenesis maybe used to introduce variations into the structure of nucleic acids.Mutations in the FIV-141 nucleic acid sequence introduced by this orsome similar method are encompassed by the invention, provided that atleast one major biological characteristic of the resulting virus, e.g.,its antigenicity, remains substantially the same as that of the virusfrom which it was derived. In a preferred though non-limitingembodiment, mutations that detectably reduce the infectivity of FIV-141compared to that of the wild-type strain fall within the scope of theinvention. For example, a specific mutation in the ENV gene is describedherein below which produces a virus that replicates, but which showsgreatly reduced infectivity. The present invention includes both thismutation and other mutations that result in substantially reduced viralinfectivity.

As discussed in the Examples section, the complete FIV-141 genome hasbeen cloned and a plasmid containing the full length sequence has beendeposited as ATCC number 203001. Standard methodology may be used toisolate this plasmid and transfect it into host cells capable ofsupporting virus production. Crandell feline kidney (CRFK) cells havebeen found to be suitable for this purpose, but other cell types suchas, e.g., feline T lymphocyte cells, may be used as well. In some cases,host cells expressing the virus may be used directly, e.g., they may beharvested and used to generate antibodies or in a vaccine.Alternatively, the virus produced in the cells may be isolated in highlypurified form using known separation techniques such as sucrose-gradientcentrifugation. Such methods are effective both for the wild type virusand for mutant forms of the virus.

An alternative method for obtaining the FIV-141 genome is to perform PCRamplification using primers corresponding to sequence elements in SEQ.ID NO:1. In general, the primers should be about 20 to 50 bases inlength. A strategy may be designed for amplifying the entire FIV-141genome or, alternatively, portions of the genome may be amplifiedseparately and then joined together to form the full length sequence.The Examples section below describes specific primers and methods thathave been found to be effective, but alternative procedures may bedeveloped and used as well.

If virus is being isolated from a natural source, then primers for PCRshould correspond to sequences unique to FIV-141. Successfulamplification using such primers will indicate that a virus responsiblefor an infection is, in fact, FIV-141. Thus, PCR may be used bothdiagnostically, as well as in methods for isolating virus.

B. Production of Inactivated Virus and Fixed Host Cells

The FIV-141 virus, or a degenerate variant thereof, may be used togenerate antibodies in an appropriate host and in vaccines. In eithercase, it will usually be desirable to inactivate the virus prior toadministering it to an animal. Inactivation may be accomplished by anymeans known in the art. For example, virus may be purified and theninactivated by incubation for about 24 hours in 0.8% formalin or 1.25%paraformaldehyde. Such procedures may be used either with wild type ormutant viruses.

Antibodies may also be generated or immunizations accomplished usinghost cells infected with FIV-141, mutated forms of the virus, ordegenerate variants thereof. Any host cell capable of supporting viralreplication may be used including peripheral blood mononuclear cells(PBMCs). Ordinarily, cells should be fixed prior to administration to ananimal. Fixing may be performed by treating cells with paraformaldehyde(e.g., 1.25%) for a period of about 24 hours at 37° C. The other methodsdiscussed above for inactivating virus may also be used for fixingcells, as may any other method disclosed in the art.

C. Production of Attenuated Virus

As discussed above, vaccines may be produced using either inactivatedvirus or fixed host cells. An alternative method is to use a virus thathas been attenuated by mutating one or more genes in the viral genome.The objective is to produce a virus that is not infectious whenadministered to a cat. Rates of viral replication can be determined invitro by infecting cells (e.g., PBMCS) with virus, and then determininghow the levels of virus change with time. Replication can be followedusing an immunological assay that measures an antigen specific for FIV,by quantitative PCR, or by measuring the activity of an enzyme specificto the virus (e.g., reverse transcriptase). Infectivity can bedetermined by exposing feline T lymphocytes (e.g., FeP2 cells) to virusand determining the extent to which the cells take up the virus andsupport replication (see Example 4 below).

Mutations in the FIV-141 genome may be made by site-directedmutagenesis. One way to carry this out is to amplify viral genes withprimers that introduce alterations into the normal gene sequence, Forexample, unique restriction sites may be introduced in a selected regionof the genome and the sequence between such sites may then be excised byrestriction enzyme digestion. After excision, the remaining portion ofgenomic DNA may be religated and then introduced into an appropriatehost cell to produce mutated virus. A detailed description of the makingand testing of mutated viruses and mutated viral genomes is provided inExamples 3 and 4 below. A summary of various mutations that have beenintroduced may be found in Table 1.

TABLE 1 Mutations in FIV-141 NUCLEOTIDES AMINO ACIDS MUTATION DELETEDDELETED MA del 123 bases, nucleotides 879-1001 41 amino acids, residues85-125 at the C- terminus of the MA protein CA del 114 bases,nucleotides 1056-1169 38 amino acids, residues 9-46 at the N- terminusof the CA protein NC del 242 bases, nucleotides 1635-1876 21 amino acidsin the CA region and 51 amino acids in the NC region, accompanied by areading frame shift preventing expression of the terminal portion of theNC protein ENV del 2103 bases deleted, nucleotides 701 amino acids fromthe middle of the 6577-8679 ENV protein, residues 106-806 SU del 1509bases, nucleotides 6577- 503 amino acids of the SU protein, 8085residues 106-108 V3/4 del 432 bases, nucleotides 7339-7770 144 aminoacids of the V3 and V4 regions of SU, residues 360-503 V7/8 del 216bases, nucleotides 8380-8595 72 amino acids from the V7 and V8 variableregions of the TM protein, residues 98-169 TMf del 75 bases, nucleotides8071-8145 25 amino acids in the cleavage junction between SU and TM CTdel 138 bases, nucleotides 8686-8823 46 amino acids from the cytoplasmicdomain of TM DU del 345 bases, nucleotides 4019-4363 115 amino acidsfrom the DU protein, residues 9-123 IN del 669 bases, nucleotides4418-5086 223 amino acids of the IN protein, residues 9-231 Vifn del 150bases, nucleotides 5286-5435 50 amino acids from the N-terminal portionof the Vif protein, residues 19-68 Vifc del 438 bases, nucleotides5436-5873 146 amino acids from the C-terminal portion of Vif, residues69-214 Vif del 588 bases, nucleotides 5286-5873 196 amino acids from theVif protein, residues 19-214 Orf(2) del 237 bases, nucleotides 5988-622479 amino acids of the ORF(2) protein RRE del 84 bases, nucleotides8827-8910 —

Examples of several specific mutations that produce attenuated virusessuitable for administration to animals to induce antibody production orfor use in vaccines are MA del, ENV del, V3/4 del, V7/8 del, TMf del, CTdel, Vif del, Vifc del, Vifn del, ORF(2) del, CA del, NC del, SU del, INdel, DU del, and RRE del. Thus, viruses mutated in any of the Vif, MA,ORF(2), ENV, Vifn, Vifc, V3/4, V7/8, TMf, CT, SU, CA, NC, IN, DU, or RREgenes are attenuated, and other nucleotide deletions or alterations thatinactivate these genes should produce viruses with similarcharacteristics.

D. Generation of Antibodies to FIV-141 and Treatment of Infected Cats

Antibodies to FIV-141 can be produced in any of the animals typicallyused for antibody production, including mice, rabbits, etc. However, itis preferred that the antibodies be produced in cats. If wild type virusis used as antigen, the virus should be inactivated prior toadministration. When attenuated viruses are used, e.g., viruses mutatedso as to reduce or eliminate their infectivity, inactivation or thefixing of host cells is not required, although these procedures may beperformed if desired.

Compositions containing virus may be administered to animals by anyroute, but animals will typically be injected intramuscularly,subcutaneously or intravenously. Generally, the virus preparation willinclude an adjuvant, e.g., Freund's complete or incomplete adjuvant.Appropriate preparations for injection, injection schedules and the likeare well known in the art and may be employed for FIV-141 and itsmutants (see, e.g, Harlow, et al., 1988, Antibodies, A LaboratoryManual, Cold Spring Harbor Laboratory, N.Y.; Klein, 1982, Immunology:The Science of Self-Nonself Discrimination). Monoclonal antibodies mayalso be used, and can be made using standard procedures (Kennett, etal., 1980, Monoclonal Antibodies and Hybridomas: A New Dimension inBiological Analyses; Campbell, 1984, “Monoclonal Antibody Technology,”in: Laboratory Techniques in Biochemistry and Molecular Biology).

Antibodies or fragments of antibodies reacting with specificity toFIV-141 (i.e., having at least a 100 fold greater affinity for FIV-141than for any other virus) may also be used in any of a variety ofimmunoassays. For example, the antibodies may be used to detect FIV-141in radioimmunoassays or in immunometric assays, also known as “2-site”or “sandwich” assays (see Chard, 1978, “An Introduction to RadioimmuneAssay and Related Techniques,” in: Laboratory Techniques in Biochemistryand Molecular Biology, North Holland Publishing Co. N.Y.). In a typicalimmunometric assay, a quantity of unlabeled antibody is bound to a solidsupport that is insoluble in the fluid being tested, e.g., blood, lymph,cellular extracts, etc. After the initial binding of antigen toimmobilized antibody, a quantity of detectably labeled second antibody(which may or may not be the same as the first) is added to permitdetection and/or quantitation of antigen (see, e.g., Kirkham et al.(ed.), 1970, Radioimmune Assay Methods, pp. 199-206, E&S Livingstone,Edinburgh). Many variations of these types of assays are known in theart and may be employed for the detection of FIV.

E. Conventional Vaccines and Vaccination Procedures

Vaccines and vaccination procedures employing different strains of FIVor closely related viruses have been discussed by a number of authors(Elyar et al., 1997, Vaccine 15:1437-1444; Yamamoto et al., 1993, J.Virol. 67:601-605; Murphey-Corb et al., 1989, Science 240:1293-1297;Jarrett et al., 1990, AIDS 4:S163-S165; and Desrosiers et al., 1989,Proc. Nat'l Acad. Sci. USA 86:6353-6357). In the case of FIV-141, thereare three types of vaccines that may be used: inactivated whole virusvaccine, fixed cell vaccines, and attenuated virus vaccines.

Typically, a vaccine will contain between about 1×10⁶ and about 1×10⁸virus particles in a volume of between about 0.5 and 5 ml. Formulationmay take place using standard methods such as those described inRemington's Pharmaceutical Sciences, 1982, Mack Publishing Co., Easton,Pa. 16th ed. Preparations may contain inactivated virus, fixed hostcells or attenuated virus, together with a pharmaceutically orveterinarily acceptable carrier as known in the art and one or moreadjuvants. Vaccines will generally be designed for parenteraladministration, although the present invention is compatible with otherforms of administration as well, such as e.g., by oral, intranasal,intramuscular, intra-lymph node, intradermal, intraperitoneal,subcutaneous, rectal or vaginal administration, or by a combination ofroutes. The skilled artisan will readily be able to formulate thevaccine composition according to the route chosen.

The most preferred vaccines will contain attenuated virus that iscompletely, or essentially completely, non-infectious when administeredto cats.

Immunization procedures will typically involve several inoculations withvaccine (e.g., 3 inoculations) separated by intervals of 3 to 10 weeks.Procedures for optimizing inoculation schedules and the other parametersassociated with immunization are well known in the art.

F. DNA Vaccines

References describing vaccines and vaccination procedures that utilizenucleic acids (DNA or mRNA) include U.S. Pat. No. 5,703,055, U.S. Pat.No. 5,580,859, U.S. Pat. No. 5,589,466, International Patent PublicationWO 98/35562, and various scientific publications, including Ramsay etal., 1997, Immunol. Cell Biol. 75:360-363; Davis, 1997, Cur. OpinionBiotech. 8:635-640; Manickan et al., 1997, Critical Rev. Immunol.17:139-154; Robinson, 1997, Vaccine 15(8):785-787; Robinson et al.,1996, AIDS Res. Hum. Retr. 12(5):455-457; Lai and Bennett, 1998,Critical Rev. Immunol. 18:449-484; and Vogel and Sarver, 1995, Clin.Microbiol. Rev. 8(3):406-410, which are incorporated herein byreference. These procedures may be utilized to produce a vaccine againstFIV in which nucleic acid corresponding to attenuated FIV-141, or adegenerate variant thereof, is administered to a cat. Immunogensdelivered in this manner typically evoke both a humoral and cytotoxicimmune response.

Either DNA or RNA encoding the attenuated FIV-141 genome, or degeneratevariant thereof, may be used in vaccines, but DNA will generally bepreferred. The DNA may be present in a “naked” form or it may beadministered together with an agent facilitating cellular uptake (e.g.,liposomes, or cationic lipids). The polynucleotide molecule may beassociated with lipids to form, e.g., DNA-lipid complexes, such asliposomes or cochleates. See, e.g., International Patent Publications WO93/24640 and WO 98/58630, which are incorporated herein by reference.

The typical route of administration will be by intramuscular injectionof between about 0.1 and 5 ml of vaccine. The total polynucleotide inthe vaccine should generally be between about 0.1 ug/ml and about 5.0mg/ml. Polynucleotides may be present as part of a suspension, solutionor emulsion, but aqueous carriers are generally preferred.

Immunization of cats using DNA vaccines may be performed as either asingle inoculation or as multiple inoculations of divided dosesseparated, e.g., by intervals of 3 to 10 weeks. If desired, serum may becollected from inoculated animals and tested for the presence ofantibodies to FIV-141 or other strains of FIV.

G. Extension of Methodology to Other Lentiviruses

The methods discussed above for attenuating FIV-141 and for producingmutated viral nucleic acids suitable for use in vaccines may be extendedto other strains of FIV and to other types of lentivirus. In each case,the viral genome is cloned and mutated in one or more genes selectedfrom the group consisting of: MA, CA, NC, DU, ENV, SU, TMf, CT, ORF(2),Vif, Vifn, Vifc, V3/4, V7/8, IN, and RRE. The mutation should bedesigned to inactivate the gene product. This can usually be most easilyaccomplished by deleting either the entire gene or a substantial portionof the gene. Although the specific methodology used for inducingmutations will vary depending upon the virus, the basic techniques areroutine in the art and, using the procedures disclosed herein as aguide, can be readily carried out by a skilled molecular biologist.Antibody production, the making and administration of vaccines, etc.,may be accomplished as discussed in connection with FIV-141 herein,making minor adaptations as needed.

In addition, it is contemplated that antibodies to certain lentivirusesmay be used to provide passive immunity to an animal or human infectedwith the virus. Either purified antibody or antibody-containing serummay be used for this purpose, and preparations may be administered on aperiodic basis until an improvement in one or more signs associated withviral infection is observed.

EXAMPLES

Example 1

Construction of an Infectious FIV Proviral DNA Clone A. Isolation andCloning of FIV-141

Virus Isolation

FIV-141 was isolated from the plasma of an FIV-infected cat. The viruswas amplified by administering plasma from the infected animal to aspecific pathogen-free (SPF) cat. Infection of the inoculated cat wasconfirmed by virus isolation and seroconversion. The cat was euthanized12 weeks post challenge, tissues were collected, and the spleen was usedas the source of virus for the molecular cloning of the FIV-141 genome.Genomic DNA was isolated from the infected spleen using a DNA extractionkit (Stratagene, La Jolla, Calif.) according to the protocol provided bythe manufacturer. Purified genomic DNA was dissolved in TE buffer at aconcentration of 1 ug/ml and stored a −70° C.

PCR Amplification and Cloning of Three Segments of the FIV-141 Genome

Three sets of oligonucleotides were designed based upon the publishedsequences of other FIV isolates (Talbott et al., 1989, Proc. Nat'l Acad.Sci. USA, 86:5743-5747; Miyazawa et al., 1991, J. Virol. 65:1572-1577;Talbott et al., 1990, J. Virol. 64:4605-4613). These oligonucleotideswere used to amplify three segments of the FIV-141 genome, one at the 5′end, one at the 3′ end and one in the middle of the genome. Because of alow copy number of the FIV proviral genome in infected tissue, tworounds of PCR amplification were performed using a semi-nested set ofprimers for each segment.

Three primers were used to clone a segment from the 5′ end of theFIV-141 proviral genome, extending from nucleotides 118 to 646. Thisregion covers most of the 5′ long terminal repeat, the interveningsequence between the 5′ long terminal repeat and the Gag open readingframe, and the N-terminal portion of the Gag gene. The sequences of theprimers were as follows: the forward primer pr-1 (117-CCGCAAAACCACATCCTATGTAAAGCTTGC-146, SEQ ID NO:2) and the two reverse primers, pr-2(646-CGCCCCTGTCCATTCCCCATGTTGCTGTAG-617, SEQ ID NO:3) and pr-8(1047-TTACTGTTTGAATAGGATATGCCTGTGGAG-1018, SEQ ID NO:4). First round PCRamplification was performed using 200 ng each of pr-1 and pr-8 asprimers and 1 ug of genomic DNA as template, with a mixture of 0.5 unitsof Taq DNA polymerase (Gibco, BRL Gaithersburg, Md.) and 1 unit of PfuDNA polymerase (Stratagene, La Jolla, Calif.) Amplification proceeded at94° C. for one minute; followed by 30 cycles of denaturing at 94° C.,for 45 seconds; annealing at 52° C. for 45 seconds; and extension at 72°C. for two minutes. The second round amplification was performed usingprimers pr-1 and pr-2 together with 2 ul of the first round PCR productsas template. The same conditions used in the first round ofamplification were applied in the second round, except that annealingtook place at a temperature of 55° C.

Three oligonucleotides were also used to clone a segment from the 3′ endof the FIV-141 proviral genome. This segment includes nucleotides 8874to 9367, consisting of most of the 3′ long terminal repeat and theintervening sequence between the 3′ long terminal repeat and the ENVgene. The sequences of the three primers were as follows: the twoforward primers, pr-5 (8793-GCAATGTGGCATGTCTGAAAAAGAGGAGGA-8822, SEQ IDNO:5) and pr-7 (8874-TCTTCCCTTTGAGGAAGATATGTCATATGAATCC-8907, SEQ IDNO:6), and the reverse primer, pr-6(9367-TCTGTGGGAGCCTCAAGGGAGAACTC-9342, SEQ ID NO:7). Primers pr-5 andpr-6 were used to perform the first round amplification, and pr-6 andpr-7 were used to carry out the second round of amplification. The sameconditions were applied to the present amplification as those used inthe amplification of the segment from the 5′ and of FIV-141 describedabove.

In order to clone a segment from the middle part of the FIV-141 genome,extending from nucleotides 5147 to 5631 and covering the C-terminalportion of the IN gene and the N-terminal portion of the Vif gene, afirst round amplification was performed using the forward primer, pr-3(4738-ACAAACAGATAATGGACCAAATTTTAAAAA4767, SEQ ID NO:8) and the reverseprimer pr-10 (5631-TTTCAATATCATCCCACATAAATCCTGT-5604, SEQ ID NO:9). Asecond round amplification was performed using forward primer pr-9(5147-TTAAAGGATGAAGAGAAGGGATATTTTCTT-5176, SEQ ID NO:10) and reverseprimer-pr-10.

After the completion of the second round of PCR amplification, theproducts were applied to a 1% agarose gel and the expected bands for allthree regions were purified by a Wizard PCR Preps kit (Promega, Madison,Wis). The purified PCR fragments were cloned into pCR-Script Amp SK(+)vectors (Stratagene, La Jolla, Calif.) according to the procedurerecommended by the manufacturer. Inserts were confirmed by restrictionenzyme digestion followed by sequencing the two strands of the plasmidDNA (Advanced Genetic Analysis Center, St. Paul, Minn.). In order toeliminate “errors” in the FIV sequences generated by the DNA polymerasesduring amplification, three clones from three independent PCRamplifications were sequenced for each region. The consensus sequencefrom the three independent clones was considered as the authenticFIV-141 sequence.

Combining the sequences from the 5′ and 3′ end segments suggests thatthe long terminal repeat of FIV-141 consists of 354 bases, including 208bases in the U3 region, 79 bases in the R region and 67 bases in the U5region. The terminal 2-base inverted repeats, the TATA box, thepolyadenylation signal, and a number of putative cis-actingenhancer-promoter elements were perfectly conserved relative to otherFIV isolates.

PCR Amplification and Cloning of the Entire Proviral Genome of FIV-141

Sequence information obtained using the three cloned segments describedabove was used to design FIV-141 specific primers that could be used toamplify and clone the entire proviral genome in two pieces, the 5′ halfand the 3′ half. Each half was amplified by two rounds of amplificationwith a semi-nested set of primers.

To amplify the 5′ half of the FIV-141 genome (from nucleotide 1 to5460), the first round of amplification was performed using forwardprimer, pr-11 (1-TGGGAAGATTATTGGGATCCTGAAGAAATA-30, SEQ ID NO:11) andthe reverse primer pr-10. The amplification protocol followed thatprovided with the Advantage Genomic PCR Kit from Clonetech (Palo Alto,Calif.). Briefly, the PCR reaction was set up in a total volume of 50ul, containing 1 ul of genomic DNA template (1 ug/ul), 1 ul of eachprimer (100 ng/ul), 5 ul of the 10×Tth PCR reaction buffer, 2.2 ul of 25mM Mg (OAc)₂, 1 ul of 50×dNTP mix (10 mM each), 1 ul of 50×Advantage TthPolymerase mix, 1 ul of Pfu polymerase (2.5 U/ul), and 36.8 ul ofsterile water. The reaction mix was heated at 94° C. for 2 minutes,followed by 30 cycles of amplification: 94° C. for 30 seconds and 68° C.for 6 minutes. The second round amplification was carried out using 2 ulof the first round PCR product as template, the same forward primerpr-11 and the reverse primer, pr-12 (5460-CATATCCTATATAATAATCACGCGTATGAAAGCTCCACCT-5421, SEQ ID NO:12). To facilitate the construction of afull length FIV-141 genome from the two halves, the restriction enzymesite Mlu I (underlined) was incorporated into the primer pr-12. The samePCR conditions as used in the first round amplification were applied inthe second round and resulted in the production of an amplificationfragment with the size of 5460 base pairs.

To clone the 3′ half of the FIV-141 proviral genome, three primers,pr-9, pr-13 and pr-14, were initially used to perform amplifications.The first round PCR amplification was carried out using forward primer,pr-9 and reverse primer, pr-14 (9464-TGCGAGGTCCCTGGCCCGGACTCC-9441, SEQID NO:13). The second round amplification was performed using forwardprimer, pr-13 (5421-AGGTGGAGCTTTCA TACGCGTGATTATTATATAGGATATG-5460, SEQID NO: 14) and the same reverse primer pr-14. Primer pr-13 was designedto overlap with pr-12 primer used in the amplification of the 5′ half ofthe genome. As in the pr-12 primer, an Mlu I restriction enzyme site(underlined) was incorporated into pr-13 to facilitate the constructionof the full-length FIV clone. Unfortunately, after two rounds of PCRamplification, no specific DNA band was observed. It was concluded thatthe failure to amplify the 3′ half of the FIV-141 genome was probablydue to the high GC content and very stable secondary structure in primerpr-14. Therefore, a new primer, pr-16(9444-CTCCAGGGATTCGCAGGTAAGAGAAATTA-9416, SEQ ID NO:15) was designed.This sequence ends 20 bases upstream of the last base in the FIV-141genome. First round PCR amplification was performed using forward primerpr-9 and reverse primer pr-16. This was followed by amplification usingforward primer pr-13 and, again, reverse primer pr-16. A DNA fragmentwith the expected size was obtained after the second roundamplification.

The DNA fragments of the 5′ half and 3′ half of the FIV-141 genome werepurified using the Wizard PCR Preps DNA purification kit (Promega,Madison, Wis.), and cloned into pCR-Script Amp SK(+) cloning vectors(Stratagene, La Jolla, Calif.). Three clones from three independent PCRreactions were sequenced for each of the 5′ half and 3′ half clones.Both strands of plasmid DNA were sequenced and the authentic consensussequence for the entire genome was obtained by comparing the resultsobtained for the three independent clones. The DNAStar program (DNAStarInc., Madison, Wis.) was used to perform sequence assembly, comparisonand analysis.

B. Molecular Characterization of the Cloned FIV-141 Virus

Sequence Results and Analysis of the Entire FIV-141 Genome

The entire proviral genome of FIV-141 was found to contain 9464 bases.The genome is organized in a manner typical of lentiviruses and consistsof: 5′ and 3′ long terminal repeats; three large open reading frames(ORF) containing the Gag, Pol, and ENV genes; and three small openreading frames containing the Vif, Rev, and ORF(2) regulatory proteins.The long terminal repeat shares 78.6% and 93.9% sequence homology withFIV-Petaluma (Olmsted et al., 1989, Proc. Nat'l Acad. Sci. USA86:2448-2452) and FIV-USIL (Sodora et al., 1995, AIDS Res. Hum.Retroviruses 11:531-533) isolates, respectively. The Gag polyproteinshares 88.4% and 94.4% amino acid homology with FIV-Petaluma andFIV-USIL isolates, respectively.

The Gag gene encodes the matrix (MA) protein (bases 627 to 1031), capsid(CA) protein (bases 1032 to 1724), and nucleocapsid (NC) protein (bases1725 to 1976). The Gag and Pol polyprotein overlap 97 bases with the PolORF, beginning at nucleotide 1880 and ending at nucleotide 5239. Aheptanucleotide frameshift signal (5′-GGGAAAC-3′) is located 100 basesupstream of the 3′ end of the overlap. As the result of a −1 frameshiftduring translation, a Gag/Pol polyprotein fusion is produced.

Compared with FIV-Petaluma and FIV-USIL isolates, the Pol polyprotein ofFIV-141 exhibits an 85.7% and 92.2% amino acid identity, respectively.The Pol gene encodes: a lead sequence from nucleotide 1880 to 1978; aprotease (PR) from nucleotide 1979 to 2326; a reverse transcriptase (RT)from nucleotide 2327 to 3994; a deoxyuridine triphosphatase (DU) fromnucleotide 3995 to 4393; and an integrase (IN) from nucleotide 4394 to5239.

The Vif ORF overlaps eight bases with the Pol gene, and shares 80.2% and91.3% amino acid homology with FIV-Petaluma and FIV-USIL isolates,respectively. Immediately following the Vif gene is the ORF(2) gene,beginning at nucleotide 5988 and ending at 6224, which evidences a 62%and 92.4% sequence homology with FIV-Petaluma and FIV-USIL isolates,respectively.

The ENV polyprotein shares a 79.3% and 88.6% amino acid identity withFIV-Petaluma and FIV-USIL isolates, respectively. The ENV gene encodes:a surface (SU) protein, from nucleotide 6262 to 8088; and atransmembrane (TM) protein, from nucleotide 8089 to 8826.

The Rev protein results from the translation of a multiple splicingmRNA. The first exon of the putative Rev gene apparently shares aninitiation codon with the ENV gene, beginning at nucleotide 6262 andending at 6505. The second Rev exon begins at nucleotide 8947, extendsinto the U3 region of the 3′ long terminal repeat, and ends atnucleotide 9161. The Rev protein of FIV-141 has a 67.3% and 83.9% aminoacid homology with FIV-Petaluma and FIV-USIL isolates, respectively. The151 base Rev responsible element (RRE) overlaps 52 bases with the ENVgene, beginning at nucleotide 8775 and ending at 8925.

Based on the sequence comparisons within the V3 region of the SUglycoprotein, FIV-141 is a type B isolate. Apparently, FIV-141 is mostclosely related to FIV-USIL, another type B FIV isolate.

C. Construction of a Full-length Molecular Clone of FIV-141

In order to construct a full-length FIV-141 clone, the 20 bases at theextreme 3′ end of the genome had to be added to the 3′ half clone. Inaddition, a consensus sequence was identified by comparing the sequencesfrom three independent clones. Site directed mutagenesis (SDM) was thenused to adjust the sequences of the 5′ and 3′ half clones to match thatof the consensus before construction of the full length viral clone.

Addition of Missing 20 Bases at the Extreme 3′ End of FIV-141

In order to add the 20 bases to the 3′ half clone of FIV-141, the longterminal repeat was first PCR amplified and cloned into a pCR-Script AmpSK(+) cloning vector using the 5′ half clone as template and forwardprimer, PR-21 (5′-TTACAAGAATTCAACTGCAGTGGGAA GATTATTGGGATCCTGAAGAAAT-3′,SEQ ID NO:16) and reverse primer, pr-20(5′-TTCAAGGAGCTCTTTTGTCGACAACTGCGAGGTCCCTGGCCC-3′, SEQ ID NO:17). Inorder to facilitate cloning the PCR fragment, two restriction enzymesites (underlined), EcoRI and Pst I, were incorporated into the forwardprimer, PR-21, and two sites (underlined), Sac I and Sal I, wereincorporated into the reverse primer, PR-20. The FIV-141 specificsequences in the primers are shown in italics. The resulting clone wassequenced and designated as pCR-LTR. A restriction fragment of pFIV-LTRgenerated by digestion with Sac I and Nhe I was cloned into one of the3′ half clones of FIV-141. The resulting clone was named pFIV3′-2A-1′and the presence of the 20 bases at the extreme 3′ end of FIV-141 wasconfirmed by nucleotide sequencing.

Construction of Consensus Sequence in the 5′ and 3′ Half Clones

In order to establish the consensus sequence in the existing 5′ and 3′half clones of the FIV-141 genome, SDM was performed. To introducesequence changes in the first half of the genome, one of the 5′ halfclones (designated as “pFIV5′-D-11”) was used as template. There were atotal of 15 nucleotide changes in pFIV5′-D-11 compared to the consensussequence. Two changes were located in the 5′ non-coding region, A602Gand A612G, and seven nucleotide changes in the coding region were silentmutations. The other six changes in the coding region resulted in anamino acid substitution for each change, three in the RT region (anA2890G nucleotide change, resulting in an I to M amino acidsubstitution; a G3461A change resulting in an E to K substitution; and aG3737A change resulting in an E to K substitution), one in the DUprotein (a C4383T change, resulting in a T to I substitution), and twoin the IN protein (an A4579G change, resulting in an I to Msubstitution; and an A5007T change, resulting in a Q to L substitution).Seven oligonucleotides were designed to make the two changes in thenon-coding region and six changes in the coding region leading to aminoacid substitutions. The oligonucleotides are as follows, with mismatchesunderlined:

Oligo pF-1, designed to repair the A602G and A612G errors: 5′-GATTCGTCGGGGGACAGCCAACAAGGTAGGAGAGATTCTACAGCAACATGGGG-3′ (SEQ ID NO:18);

Oligo pF-2, designed to fix the error A2890G: 5′-TCMTATATGGATGATATCTATATAGGATCAAATTTAAGTAA-3′ (SEQ ID NO:19);

Oligo pF-3, designed to repair the error G3461A: 5′-GTGATATAGCTCTAAGGGCATGTTACAAAATAAGAGAAGAATCCATTATAAGAATAGG-3′ (SEQ ID NO:20);

Oligo pF-4 was designed to repair the error G3737A: 5′-CGGGCAGATGGCAGGTAATGGAAATAGAAGGAGTAATCAAAAAGC-3′ (SEQ ID NO:21);

Oligo pF-5, designed to repair the error C4383T: 5′-AGAAAGGGATTTGGGTCAACTGGAGTCTTTTCTTCATGGGTGGA-3′ (SEQ ID NO:22);

Oligo pF-6, designed to repair the error A4579G: 5′-GGGGGACAATTAAAGATTGGACCTGGCATATGGCAAATGGACTGTACACAC-3′ (SEQ ID NO: 23); and

Oligo pF-7, designed to repair the error A5007T: 5′-GGCTCCTTATGAATTATACATACAACAGGAATCATTAAGAATACAAGAC-3′ (SEQ ID NO:24).

In order to make sequence changes in the 3′ half of the genome, the 3′half clone “pFIV3′-2A-1⁺” was used as a template in performing SDM.There were nine changes in the pFIV3′-2A-1⁺ clone compared to theconsensus sequence. Two nucleotide changes in the coding region weresilent. The other seven changes all resulted in an amino acidsubstitution: one in the Vif protein (T5508C, H to Y); one in the ORF(2)region (A6041T, D to E); three in the SU protein (A6922G, V to I;G7007T, T to R; and A7814G, S to N); one in the TM region (A8405T, I toN); and one in the Rev region (A8976G, E to K). Seven mutagenesisoligonucleotides were designed for repairing these seven amino acidsubstitutions:

Oligo pF-8, designed to repair the error T5508C: 5′-CAAAATAGTTTAAGATTGTATGTTTATATAAGCAAT-3′ (SEQ ID NO:25);

Oligo pF-9, designed to repair the error A6041T: 5′-CAGAAAAGTTAGATAGAGAAGCAGCTATTAGATTGTTTAT-3′ (SEQ ID NO:26);

Oligo pF-10, designed to repair the error A6922G: 5′-TAAAAGCAAATGTTAATATAAGTATACAAGAAGGACCTAC-3′ (SEQ ID NO:27);

Oligo pF-11, designed to repair the error G7007T:5′-AAAAGCTACAAGGCAATGCAGAAGGGGAAGGATATGGAAG-3′ (SEQ ID NO:28);

Oligo pF-12, designed to repair the error A7814G: 5′-AGAGGACCTTATTGTACAATTTAATATGACAAAAGCAGTGGAAA-3′ (SEQ ID NO:29);

Oligo pF-13, designed to repair the error A8405T: 5′-CCCTCAATCTGTGGACAATGTATAACATGACTATAAATCA-3′ (SEQ ID NO:30);

Oligo pF-14, designed to repair the error A8976G: 5′-GACAACGCAGAAGAAGAAAGAAGAAGGCCTTCAAAAAATT-3′ (SEQ ID NO:31).

Single stranded DNA template preparations for both clones, pFIV5′-D-11and pFIV3′-2A-1⁺, were made essentially by the protocol provided by themanufacturer (Promega, Madison, Wis.). Briefly, DNA plasmids of the twoclones were transfected into the E. coli strain CJ236. Single stranded(SS) DNA was rescued using helper phage R408 and purified byphenol/chloroform extraction. Purified SS DNA was dissolved in TE bufferwith its concentration being estimated by running 2 ul samples oftemplate preparations on a 1% agarose gel. Oligonucleotides werephosphorylated according to the protocol provided by the manufacturer(Gibco BRL, Gaithersburg, Md.).

Oligonucleotides were annealed to template in a total reaction volume of30 ul. This contained 0.2 pmol of single-stranded DNA template(pFIV5′-D-11 or pFIV3′-2-A-1⁺), 4 pmol of each oligonucleotide (i.e.,pF-1 to pF-7 for the pFIV5′-D-11 template and pF-8 to pF-14 for thepFIV3′-2-A-1⁺ template), 3 ul of annealing buffer (0.2 M Tris-HCl, pH7.4, 20 mM MgCl₂, and 0.5 M NaCl). The mixture was incubated at 85° C.for 5 minutes and then gradually cooled to room temperature at a rate ofapproximately 1° C. per minute.

In order to synthesize the complementary DNA strand, the followingcomponents were added to the annealing mixture: 3 ul of synthesis buffer(4 mM of each dNTP, 7.5 mM ATP, 175 mM Tris-HCl, pH 7.4, 37.5 mM MgCl₂,215 mM DTT), 3 ul of T4 DNA ligase (3 U/ul), and 3 ul of the diluted T7DNA polymerase (0.5 units per ul). The reaction was incubated at 37° C.for 3 hours, followed by heat inactivation at 68° C. for 10 minutes. Twoul of the SDM reaction mixture were used to transfect E. coli DHα-5competent cells.

For the 5′ half clone, pFIV5′-D-11, incorporation of one of themutagenesis oligonucleotides, PF-5, resulted in an addition of a Hinc IIsite. A preliminary screening with Hinc II resulted in theidentification of four positive mutants that were designated aspFIV5′-D-11/M4, pFIV5′-D-11/M-22, pFIV5′-D-11/M-28 and pFIV5′-D-11/M-52.These four mu were completely sequenced to verify the incorporation ofthe other seven desired mutations. The sequencing results revealed thatthree of the four clones contained all eight mutations. One clone,pFIV5′-D-11/M-28, had only seven positions that were mutated.

Clone pFIV5′-D-11/M-52 was selected as the 5′ half clone to be used toconstruct the full length FIV-141 clone. For the 3′ half clone,pFIV3′-2-A-1⁺, preliminary screening with BspH I digestion identified 10mutants in which a BspH I restriction site was eliminated due to theincorporation of the mutagenesis oligonucleotide pF13. Completesequencing revealed that 8 of the 10 clones contained mutations at allseven desired positions. One of the mutants, “pFIV3′-2-A-1⁺/M-21”contained all of the 8 changes and was selected to be used as the 3′half clone in constructing the full length FIV-141 clone.

Construction of the Full Length FIV-141 Clone

In order to construct the full length FIV-141 clone, a 5.5 kb Mlul/Xholfragment derived from the 5′ half clone, pFIV 5′-D-11/M-52, was ligatedto the 3′ half clone pFIV3′-2-A-1⁺/M-21, which had been digested withthe same two restriction enzymes. The full length ligation product wasscreened by PCR amplification using a forward primer directed to the 5′half clone and a reverse primer directed to the 3′ half clone. Theforward primer, pr-9, had the sequence:5′-(5147)-TTAAAGGATGAAGAGAAGGGATATTTTCTT-(5176)-3′ (SEQ ID NO:10). Thereverse primer, pr-10, had the sequence:5′-(5631)-TTTCAATATCATCCCACATAAATCCTGT-(5604)3′, (SEQ ID NO:9). Positiveclones were confirmed by restriction digestion and sequencing. One ofthe resulting full length clones was designated as “pFIV-141-B1” andselected for characterization both in vitro and in vivo.

Example 2 Demonstration That the Full Length Molecular Clone isInfectious

Transfection

Crandell feline kidney (CRFK) cells were grown in six well plates to aconfluency of 40 to 60%. Transfection was accomplished by introducing 2ug of plasmid DNA and following the basic protocol recommended by TransIT Polyamine Transfection Reagents (Mirus, Madison, Wis.). Briefly, 10ul of Trans IT Lt-1 (Panvera) was mixed with 1 ml RPMI 1640 medium andincubated at room temperature for 15 minutes. Two ug of plasmid DNA wasadded to the RPMI/Lt-1 solution and incubated for another 15 minutes atroom temperature. Media was removed from wells, cells were washed oncewith PBS, and the DNA cocktails were added to the cell monolayers. Afterincubation at 37° C. in a CO₂ incubator for four hours, the DNAcocktails were removed from the wells and 2 ml of RPMI 1650 mediumsupplemented with 3% fetal serum (FS) was added to each well.Twenty-four hours post-transfection, cell supernatants were assayed forFIV production, reverse transcriptase (RT) activity and viralinfectivity.

FIV Production From Transfected CRFK Cells

Supernatants from the transfected CRFK cells were harvested on a dailybasis after the transfection, and were assayed for FIV capsid proteinproduction using the FIV Antigen Test Kit (IDEXX, Portland, Me.),according to the protocol recommended by the manufacturer. This enzymeimmunoassay was designed to detect the predominant group-associatedantigen of FIV p26 capsid protein. FIV antigen p26 was detected at 24hours post-transfection (PT), reached a peak at 72 hours PT, and thendecreased to background levels at 11 days PT (see FIG. 1).

In order to confirm virus production from the transfected (CRFK) cells,a reverse transcriptase (RT) activity assay (Boehringer Mannheim,Indianapolis, Ind.) was performed to detect virion-associated RTactivity in the transfected cell supernatants. Briefly, 200 ul of cellsupernatant was harvested and spun 5 minutes in a microfuge to pelletcells and cell debris. Supernatants were centrifuged at 20,000 g for 20minutes at 4° C. in a swinging bucket rotor to pellet FIV virusparticles. Viral particle pellets were resuspended in 40 ul of lysisbuffer from the kit, and assays were then performed as recommended bythe manufacturer. Virus production from transfected cells wasdemonstrated in cell supernatants 48 hours post-transfection (PT).

Infection of CRFK Cells

FIV-141 wild type virus does not infect CRFK cells. In order todetermine whether the molecular clone virus exhibits similar behavior,CRFK cells were grown in 6 well plates and inoculated with 200 ul ofp26+ conditioned medium from transfected CRFK cells. After incubationfor 2 hours at 37° C., cells were washed once with PBS, and 2 ml of RPMI1640 medium supplemented with 3% FS was added to each well. Supernatantswere then monitored for virus production by FIV p26 ELISA every 3 to 4days post-transfection. It was found that, similar to the wild typevirus, the FIV-141 clone does not infect CRFK cells.

Infection of FeP2 Cells by Co-Culture With Transfected CRFK Cells

CRFK cells were grown in 6 well plates and transfected as describedabove. At 48 hours post-transfection, 2×10⁶ FeP2 cells were added toeach p26+ transfected well. After co-culture of cells for 72 hours, FeP2cells (nonadherent) were separated from CRFK cells (adherent). Thesupernatants from the FeP2 cells were harvested and monitored for virusproduction by FIV p26 ELISA every 3 to 4 days. Four days postco-cultivation, high levels of virus production were demonstrated in theFeP2 cell supernatants (see FIG. 2). Virus titer reached a plateau 6days post-transfection, indicating that the FIV-141 molecular clonevirus is infectious in FeP2 cells. The results also suggested thatinfection of CRFK cells is blocked in an early stage of virus infection,i.e, at the time of the entry of virus into cells.

Overall, it was concluded that, upon transfection into CRFK cells, theFIV-141 molecular clone can replicate in the cell, and virus particlesreleased from the cells are infectious for FeP2 T lymphocytes.

Infection of FeP2 Cells by Adsorption

FeP2 cells (2×10⁶) were suspended in 200 ul of p26+ conditioned mediumobtained from transfected CRFK cells and incubated at 37° C. for twohours. Cells were washed with PBS, suspended in 2 ml Opti-MEM mediumsupplemented with 10% heat inactivated FCS, and incubated at 37° C.Supernatants were harvested and monitored for virus production by FIVp26 ELISA every three to four days. Four days post-infection, virusrelease from infected FeP2 cells was detected in the supernatants andreached a peak by three weeks post-infection (FIG. 3). The resultsindicate that productive infection of FeP2 cells by FIV-141 can beachieved through either adsorption or co-culture with transfected CRFKcells. Compared to infection by co-cultivation, virus production reacheda plateau much more slowly when infection took place by adsorption(FIGS. 2 and 3).

Example 3 Mutant FIV-141 Clones and Their Use in Vaccines

In order to develop FIV-141 vaccine candidates, the infectious FIV-141wild-type clone was used to construct a number of gene-deleted clones.The general criteria for making the mutant clones are:

1. The deletions or mutations introduced into the FIV-141 genome must besevere enough so that virus infectivity is substantially reduced(attenuated) after the clones are transfected into cultured cells invitro or administered to cats in vivo.

2. The deletions or mutations introduced into the FIV-141 genome shouldnot abolish the replication competency of the viral genome, or theability to express viral proteins at high levels.

Other factors to be considered are whether the gene-deleted genome willintegrate into host chromosomes, whether defective virus particles willform, and the level of viral structural proteins that will be expressed.Based upon these considerations, a number of genes and elements weretargeted for deletion. Because viral genome replication was to bemaintained, neither the RT nor PR genes of FIV-141 were mutated.

A. Deletions in the Gag Region

The Gag polyprotein contains three virion structural proteins, MA, CAand NC. Three gene-deletion clones were constructed with a deletion ineach of these proteins.

MA del Mutation

Site-directed mutagenesis was performed to create two Spe I restrictionsites in the C-terminal portion of the MA protein using the 5′ halfclone pFIV5′-D-11/M-52 as template and the mutagenesis primers: Mpma-1(5′-AGTAAAGAAATTGACATGGCGATTACTAGTTTAA AAGTTTTTGCAGTGGC-3′, (SEQ IDNO:32); and Mpma-2 (5′-CCATCTATAAAAGAAAGTGGGACTAGTGAAGAAGGACCTCCACAGGC-3′, SEQ ID NO:33).

The Spe I sites introduced by the primers are underlined in thesequences. SDM was performed as discussed previously in order to repairthe putative errors in the 5′ and 3′ half clones. Mutants were screenedby Spe I restriction digestion and positive clones were used toconstruct the deletion clone. Spe I digestion was performed to releasethe Spe I fragment and the remaining part of the clone was self ligatedto create the deletion clones. These were screened by PCR amplificationusing primer sets flanking the deleted region and confirmation wasobtained by nucleotide sequencing. The 5′ half clone with the deletionwas ligated to the 3′ half clone pFIV3′-2A-1⁺/M-21 to generate the fulllength clone with the deletion. This clone was named FIV-141 MA deletionclone and contained a deletion of 123 bases from nucleotide 879 tonucleotide 1001, corresponding to 41 amino acids from residues 85 to 125at the C-terminus of MA.

CA del Mutation

SDM was performed to create two Spe I sites in the CA region of FIV-141using the 5′ half clone pFIV5′-D-11/M-52 as template and, as primers,Mpca-1 (5′-ATTCAAACAGTAAAT GGAGCAACTAGTTATGTAGCCCTTGATCCAAAAATG-3′, SEQID NO:34) and Mpca-2(5′-ACAGCCTTTTCAGCTAATTTAACTAGTACTGATATGGCTACATTAATTATG-3′, SEQ IDNO:35). After deletion of the Spe I restriction fragment, the 5′ halfclone was ligated to pFIV3′-2A-1⁺/M-21 to generate the gene-deleted fulllength clone. This clone has a 114 base pair deletion from nucleotides1056 to 1169, corresponding to the 38 amino acids from positions 9 to 46at the N-terminus of the CA protein.

NC del Mutation

The 5′ half clone has unique Sca I and Sma I sites at nucleotides 1635and 1876 respectively. The clone was digested with Sca I and Sma I torelease a 242 base pair fragment. The remaining portion of the 5′ halfclone was self-ligated and then joined to the 3′ half clone to generatethe gene-deleted full length clone. The deletion consists of 63 bases(21 amino acid residues) in the CA region, 27 bases (9 amino acidresidues) between the CA and NC protein and 152 bases (51 amino acidresidues) at the N-terminal portion of the NC protein. The deletion alsocaused a −1 reading frame shift and, therefore, the C-terminal portionof the NC protein cannot be expressed by this clone.

B. Deletions in the ENV Region

The ENV precursor glycoprotein is processed into two mature proteins: SUand TM. Six deletion clones were constructed in the ENV region.

ENV del Mutation

SDM was performed to create two BstE II sites in the ENV region usingthe 3′ half clone pFIV3′-2A-1⁺/M-21 as template and, as mutagenesisprimers, Mpenv-1(5′-ACTATAGTCTATTTACTAACTGGTTACCTGAGATATTTAATAAGCCATAG-3′, SEQ ID NO:36)and Mpenv-2 (5′-TACTTATATGCTTGCCTACATTGGGTTACCGTATAAGAAACTGTACTAATAAAA-3′, SEQ ID NO:37). The BstE II sites in the primer areunderlined. After deletion of the BstE II fragment, the self-ligatedclone was joined to pFIV5′-D-11/M-52. The resulting clone has a deletionof 2103 bases from nucleotides 6577 to 8679, corresponding to the middle701 amino acids of the ENV protein (residues 106 to 806). The N-terminal105 residues, the majority of which overlaps the first exon of the Revprotein, and the C-terminal 45 residues which overlaps with the Revresponsible element (RRE), were maintained in the deletion clone.

SU del Mutation

Two Spe I sites were generated in the SU region of FIV-141 by SDM usingclone pFIV3′-2A-1⁺/M-21 as template and, as mutagenesis primers, Mpsu-1(5′-GAGGTATAAAGG TAAACAAAAAACTAGTGCCATTCATATTATGTTAGCCCTTGC-3′, SEQ IDNO:38) and Mpsu-2(5′-ACTAACTATAGTCTATTTACTAACAACTAGTTTGAGATATTTAATAAGCCAT AGAAAC-3′, SEQID NO:39). The Spe I fragment was deleted by Spe I digestion followed byself ligation of the large remaining fragment. The resulting clone wasligated to pFIV5′-D-11/M-52. This clone has a deletion of 1509 basisfrom nucleotides 6577 to 8085, corresponding to a deletion of 503 aminoacids (residues 106 to 608) of the SU protein. The clone maintains theN-terminal 105 amino acids of the SU protein.

V3/4 del Mutation

SDM was performed to create two Sph I sites flanking the V3 and V4region of the SU protein. The clone pFIV3′-2A-1⁺/M-21 was used astemplate along with the mutagenesis primers: Mpenv-5(5′-ATACCGAAATGTGGATGGTGGAATCAGGCATGCTATTATAAT AATTGTAAATGGGAAGAAGC-3′,SEQ ID NO:40) and Mpenv-6 (5′-GCACTATGTACAATTGTTCCTTACAGGCATGCTTCACTATGAAAATAGAGGACCTTAT-3′, SEQ ID NO:41). Sph Isites are underlined. After digestion to remove the Sph I fragment, theclone was self-ligated and then joined to pFIV5′-D-11/M-52. This clonecontains a deletion of 432 bases from position 7339 to 7770,corresponding to a deletion of 144 amino acids (from residue 360 to 503)of the SU protein, covering the V3 and V4 regions.

V7/8 del Mutation

SDM was used to create two Sph I sites flanking the V7 and V8 region ofthe TM protein. This was accomplished using the clone pFIV3′-2A-1⁺/M-21as template and the mutagenesis primers Mpenv-7(5′-GAATCAATTCTTTTGTAAGATCGCATGC AATCTGTGGACAATGTATAACATGACTA-3′, SEQ IDNO:42) and Mpenv-8(5′-GGGAAAATTGGGTGGGATGGATAGGTAAGATCGCATGCTATTTAAAAGGACTTCTTGGT AG-3′,SEQ ID NO:43). Sph I sites are underlined. Digestion with Sph I resultedin the elimination of 216 bases from nucleotides 8380 to 8595, and wasfollowed by ligation of the large fragment. The resulting clone was thenjoined to the 5′ half clone pFIV5′-D-11/M-52 to generate “V7/8 del.”This contains the deletion of 72 amino acids (from residues 98 to 169)of the TM protein covering the V7 and V8 various regions.

TMf del Mutation

The 3′ half clone of FIV-141 has a unique Age I site at nucleotide 8145.SDM was performed to create a second Age I site at position 8071. The 3′half clone was used as template along with the mutagenesis primer,Mpenv-3 (5′-GGAAGAAGTTATGAG GTATACCGGTAAACAAAAAAGGGCC-3′, SEQ ID NO:44).A 75-base fragment between the two Age I sites was deleted byrestriction enzyme digestion followed by self-ligation of the largerestriction fragment. The resulting clone was ligated to the 5′ halfclone to generate “TMf del.” This contains a deletion of 25 amino acidsin the cleavage junction between the SU and TM proteins. The deletedamino acids include 6 C-terminal residues of the SU protein (4 of whichare basic (either K or R) and required for the processing of the SU/TMcleavage site) and 19 N-terminal residues of the fusion peptide of theTM protein (required for membrane fusion between virion envelope andcell membrane).

CT del Mutation

SDM was performed to truncate the cytoplasmic tail of the TM proteinusing the 3′ half clone pFIV3′-2A-1⁺/M-21 as template and themutagenesis primer, Mpenv-4(5′-CTACTTATATGCTTGCCTACATTGGTCGACTGATAGTGAAACTGTACTAATAAAATATTGG G-3′,SEQ ID NO:45). A Sal I restriction site (underlined) was incorporatedinto the oligonucleotides by silent mutation to facilitate the screeningof mutants. Three tandemly repeated translation stop codons (italicized)were incorporated in the primer right after the transmembrane domain ofthe TM protein. The resulting clone was ligated to the 5′ half clone togenerate “CT del.” This has a 138-base truncation from nucleotides 8686to 8823 (corresponding to a truncation of 46 amino acids from thecytoplasmic domain of the TM protein).

C. Deletions in the Pol Region

The Pol polyprotein consists of four enzymatic proteins: PR, RT, DU, andIN. Two deletion clones were constructed in the Pol region.

DU del Mutation

SDM was performed to create two Spe I sites in the DU region using the5′ half clone pFIV5′-D-11/M-52 as template and, as mutagenesis primers,Mpdu-1 (5′-GATGGTTATAGA AGGTGAAGGMTTACTAGTAAAAGATCAGAAGATGCAGGATATG-3′,SEQ ID NO. 46) and Mpdu-2(5′-GAAATAATAATGGATTCAGAAAGAGGAACTAGTGGATTTGGGTCAACTGGA GTCTTTTC-3′, SEQID NO:47). Spe I sites in the primers are underlined. A deletion of 345bases from nucleotide 4019 to 4363 was achieved by Spe I digestionfollowed by self-ligation of the large restriction fragment. Theresulting clone was joined to the clone pFIV3′-2A-1⁺/M-21 to generate“DU del.” The clone contains a deletion of 115 amino acids correspondingto almost the entire DU protein.

IN del Mutation

Two Spe I sites were created by SDM in the IN region of FIV-141 usingpFIV5′-D-11/M-52 as template and the mutagenesis oligonucleotides,Mpin-1 (5′-CTTCATGGGTGGACAGAATTGAAACTAGTGTATTAAATCATGAAAAATTTCACTCAG-3′,SEQ ID NO:48) and Mpin-2 (5′-GCAATGGGTGTATTATAAAGATCAGACTAGTAAAAAGTGGAAGGGACCAATGAGAGTAG-3′, SEQ ID NO:49). Spe I sites in the primersare underlined. After deletion of the Spe I fragment and self ligation,the resulting clone was joined with pFIV3′-2A-1⁺/M-21 to generate “INdel.” This contains a deletion of 669 bases from nucleotide 4418 to 5036corresponding to almost the entire IN protein (223 amino acids, fromresidue 9 to 231 of IN).

D. Deletions in Regulatory Genes or Elements

Three regulatory proteins identified in FIV are Rev, Vif and ORF2. ARev-responsible element has been reported at the 3′ end of the FIVgenome. Five deletion clones were constructed in these regions.

Vifn del Mutation

A unique Mlu I site was introduced in the middle of the Vif gene in the5′ half clone pFIV 5′-D-11/M-52. SDM was performed to create a secondMlu I site at the N-terminus of the Vif gene using pFIV3′-D-11/M-52 astemplate and the mutagenesis primer, Mpvif-1 (5′-AGAAGACTCTTTGCAGTTCTCCAATGAACGCGTTAGAGTGCCATGTTATACATATCG-3′, SEQ IDNO:50). The Mlu I site in the primer is underlined. A restrictionfragment of 150 bases from nucleotide 5286 to 5435 was deleted by Mlu Idigestion followed by self ligation of the large fragment. The resultingdeletion clone was joined to pFIV3′-2A-1⁺/M-21 to generate “Vifn del.”This contains a deletion of 50 amino acids (from residue 19 to 68) atthe N-terminal portion of the Vif protein.

Vifc del Mutation

The 3′ half clone pFIV3′-2A-1⁺/M-21 has a unique Mlu I site within theVif region. A second Mlu I site was created by SDM at the C-terminus ofthe Vif protein using pFIV3′-2A-1⁺/M-21 as template and the mutagenesisprimer Mpvif-2 (5′-CGTGTGGCAAAGAGGC TAAAACGCGTAGAGGCTGTTGTAATCAG-3′, SEQID NO:51). The Mlu I site in the primer is underlined. After deletion ofthe Mlu I fragment, the resulting clone was ligated to the 5′ half clonepFIV5′-D-11/M-52 to generate “Vifc del.” This contains a deletion of 438bases from nucleotide 5436 to 5873, corresponding to a deletion of 146amino acids (from residue 69 to 214) at the C-terminal portion of theVif protein.

Vif del Mutation

To construct “Vif del,” the Xho I/Mlu I restriction fragment of Vifc delwas replaced with a 5.3 kb Xho I/Mlu I fragment of Vifn del. Theresulting clone contains a deletion of 588 bases from nucleotide 5286 to5873, corresponding to a deletion of 196 amino acids (from residue 19 to214), almost the entire Vif protein.

ORF(2) del Mutation

Two Mlu I sites were created by SDM at nucleotides 5988 and 6224 (at theN- and C-termini of ORF(2)). This was accomplished usingpFIV3′-2A-1⁺/M-21 as template and, as mutagenesis primers, Mporf-1(5′-GTGGACGGGAGAATTATGAACGCGTGAACTAATC CCACTGTTTAATAAGGTTACAG-3′, SEQ IDNO:52) and Mporf-2 (5′-CTACATTATCCATAAATACTGCCTAGACGCGTTTCTTTTAATATTTCATCTGCAG-3′, SEQ ID NO: 53). Mlu Isites in the primers are underlined. In addition to the two Mlu I sitescreated by SDM, there is an Mlu I site at nucleotide 5436 in the clone.To construct “ORF(2) del,” a 5.4 kb Mlu I/Xho I fragment from the 5′half clone pFIV5′-D-11/M-52 was ligated to the large Mlu I/Xho Ifragment of the 3′ half clone pFIV3′-2A-1⁺/M-21. A 552 base Mlu Ifragment from position 5436 to 5988 was then inserted into the resultingclone. ORF(2) del contains a deletion of 237 bases, covering the entireORF(2) gene.

RRE del Mutation

SDM was performed to create two Spe I sites in the RRE region usingpFIV3′-2A-1⁺/M-21 as template and the mutagenesis primers, Mprre-1(5′-GGCATATCTGAA AAAGAGGAGGAATGAACTAGTATATCAGACCTGTAGAATACA-3′, SEQ IDNO:54) and Mprre-2(5′-GAGGAGGATGTGTCATATGAATCAAATACTAGTCAAAAATAACAGTAAAATCT ATATTG-3′, SEQID NO:55). Spe I sites in the primers are underlined. Deletion of theSpe I fragment was achieved by Spe I digestion followed by self ligationof the large fragment. The resulting deletion clone was ligated topFIV5′-D-11/M-52 to generate “RRE del.” This contains a deletion of 84bases from nucleotide 8827 to 8910.

E. Double Deletions

FIV-141 MA del clone was digested with Xho I and BstE II, and a 4.8 kbDNA fragment containing the first half of the FIV-141 genome waspurified and used for the construction of the three double deletionclones, MA del/TMf del, MA del/V3/4 del, and MA de/Vif del, as follows.

MA del/Tmf del Mutation

FIV-141 Tmf del clone was digested with the same two restriction enzymesand a 7.8 kb fragment, which contains the second half of the FIV-141,was isolated and purified. Ligation of the 4.8 kb and 7.8 kb fragmentsresulted in a double deletion clone, i.e., FIV-141 MA del/Tmf del, whichconsists of deletions of 41 amino acids at the C-terminus of the MA and25 amino acids in the fusion peptide of TM.

MA del/V3/4 del Mutation

FIV-141 V3/4 del clone was digested with Xho I and BstE II, and a 7.8 kbfragment comprising the second half of the FIV-141 genome was purifiedand ligated to the 4.8 kb fragment derived from the FIV-141 MA delclone. The resulting double deletion clone, i.e., FIV-141 MA del/V3/4del, contains a deletion of 41 amino acids at the C-terminus of MA and adeletion of 144 amino acids of the V3 and V4 regions within the ENV.

MA del/Vif del Mutation

FIV-141 Vif del clone was digested with Xho I and BstE II and a 7.8 kbfragment containing the second half of the genome was purified andligated to the same 4.8 kb fragment derived from the FIV-141 MA delclone. The resulting double deletion clone, i.e., FIV-141 MA del/Vifdel, has a deletion of 41 amino acids at the C-terminus of MA and adeletion of 196 amino acids of Vif.

ENV del/IN del Mutation

Plasmid DNA comprising the FIV-141 ENV del clone prepared as above wasdigested with Mlu I and Sal I, and a 2 kb fragment containing the secondhalf of the FIV-141 genome with a deletion of 2.1 kb of the ENV gene wasisolated and purified. FIV-141 IN del clone prepared as above wasdigested with the same two restriction enzymes, and a 4.7 kb fragmentconsisting of the first half of the FIV-141 genome with a deletion ofthe IN gene was purified. The two fragments were ligated and cloned intothe pCR-Script Amp SK(+) vector. The resulting double deletion clone,FIV-141 ENV del/IN del, contains a deletion of 2103 bases in the ENVgene and a deletion of 669 bases in the IN gene.

Example 4 Characterization of the FIV-141 Gene Deletion Clones A. ViralProtein Expression and/or Defective Virus Production

Each plasmid of the deletion clones was transfected into CRFK cells aspreviously described. FIV p26 ELISA assays were performed to detectviral protein expression and/or virus particle production in thetransfected cell supernatants. At 48 hours post-transfection, samplesfrom 13 of the constructs were found to produce a strong positive signalcomparable to that observed for the wild type FIV-141 molecular clone(see FIG. 4). The highest levels of virus particle production wereobserved for the six deletion clones in the ENV region, including ENVdel, TMf del, SU del, CT del, V3/4 del and V7/8 del.

Comparable levels of virus particle production were obtained for sevenother deletion clones, including three deletion clones in the Vif region(Vifn del, Vifc del and Vif del), MA del, DU del, IN del and ORF(2) del.The results indicate that the deletions carried by these 13 clones donot interfere with the formation and release of virus particles from thetransfected cells. A relatively weak positive signal was detected for NCdel, indicating that deletion in this region affects virus particleassembly or release.

No virus particle production was detected in the supernatants of cellstransfected with CA del or with RRE del. The deletion in the C-terminusof the CA protein may either abolish virus particle formation or resultin loss of the epitope recognized by the monoclonal antibody (MAb) usedin the p26 ELISA kit. As expected, deletion in the RRE region resultedin a block of the export of unspliced viral RNA from the nucleus to thecytoplasm, leading to either a total lack of, or a dramatic decrease in,the expression of viral structural proteins.

B. Intracellular RT-PCR to Detect Viral RNA Expression

Intracellular RT-PCR was performed to detect viral RNA expression in thetwo deletion clones, CA del and RRE del. Plasmid DNA for each clone wastransfected into different CRFK cells. Forty-eight hours aftertransfection, total RNA was isolated from the transfected cells using anRNeasy kit (Qiagen, Chatsworth, Calif.). The RNA was eluted in 50 ul ofDEPC water, and 2 ul of each RNA sample was used to synthesize the firststrand of cDNA using Superscript II (Gibco BRL, Gaithersburg, Md.).

A 585 base pair fragment from nucleotides 2958 to 3542 was amplifiedusing as a forward primer Sp-8 (5′-TATTATGGTGGGGATTTGAAAC-3′, SEQ IDNO:56) and, as a reverse primer, Sp-20 (5′-TAATTAGATTTGATTCCCAGGC-3′,SEQ ID NO:57). Two ul of cDNA from each reaction and, as a control, 2 ulof total RNA from each preparation, were used as the template in PCRreactions. Each reaction was performed in a volume of 100 ul using a PCRamplification kit (Gibco BRL, Gaithersburg). The reaction proceeded asfollows: 25 cycles at 94° C. for 30 seconds; 55° C. for 30 seconds; and72° C. for another 30 seconds. Ten ul from each reaction was loaded on a1% agarose gel. A specific band with the expected size was observed forboth CA del and RRE del clones, indicating that viral RNA expressionoccurred in the cells transfected with these clones. The results suggestthat the failure to detect p26 protein expression by ELISA for CA del isprobably due to either a failure of virus particle formation or a lackof the epitope recognized by the antibody used in the p26 ELISA assay.For the RRE deletion clone, viral gene expression was demonstrated byintracellular RT-PCR, but no p26 protein expression was detected usingthe ELISA assay. The discrepancy may reflect a much higher sensitivityof RT-PCR assay when compared to the ELISA.

C. Encapsidation of Viral Genome and RT Enzyme in the Defective VirusParticles

Transfection of CRFK cells by the majority of the FIV-141 deletionclones resulted in the production and release of defective virusparticles. In order to determine whether gene-deleted viral genomes andRT protein were encapsidated into virus particles, virion-associated RTPCR and RT activity assays were performed. Briefly, 48 hourspost-transfection, 200 ul of the supernatant from each transfected CRFKculture were harvested and spun 5 minutes in a microfuge to pellet cellsand cellular debris. The virus particles in the supernatants werepelleted by ultracentrifugation at 20,000 g for 20 minutes at 4° C. in aswinging bucket rotor. To test for encapsidation, virus pellets wereresuspended in 350 ul RLT buffer from the RNeasy kit, and viral RNA waspurified by elution in 50 ul DEPC water as recommended by themanufacturer. First strand cDNA was made using Superscript II, and PCRamplification was performed as described previously using the Sp-8 andSp-20 primer set.

Fourteen of 16 deletion clones showed a specific band after RT-PCRamplification, indicating that transfection of CRFK cells by theseclones produced defective virus particles and that the gene-deletedviral genomes were encapsidated. The 14 clones consisted of 6 clonesfrom the ENV region (including ENV del, TMf del, SU del, CT del, V3/4del, and V7/8 del); 3 clones from the Vif region (Vifn del, Vifc del,and Vif del); 2 clones from the Pol region (DU del and IN del); 2 clonesfrom the regulatory gene/element region (ORF(2) del and RRE del); and 1clone from the Gag region (MA del). Consistent with the p26 ELISA data,CA del showed a negative signal in the virion-associated RT-PCR assay.The NC protein is required for the packaging of the viral genome intothe virion and, as expected, no virion-associated gene-deleted viral RNAgenome was detected by RT-PCR for NC del. For RRE del, virion-associatedgene-deleted viral RNA was demonstrated to be present but no virusparticle production was detected using the p26 ELISA assay. Thediscrepancy in these results may again reflect a much higher sensitivityof the RT-PCR assay relative to the ELISA.

For testing encapsidation of the RT (i.e. reverse transcriptase) enzymein defective virus particles, virus pellets were resuspended in 40 ul ofthe lysis buffer from the RT ELISA kit, and the assay was allowed toproceed as recommended by the manufacturer. Consistent with dataobtained using p26 ELISA assays and virion-associated RT-PCR assays,virion-associated RT activity could be detected for 14 deletion clones,including ENV del; SU del; TMf del; V3/4 del; V7/8 del; CT del; MA del;DU del; IN del; Vifn del; Vifc del; Vif del; ORF(2) del; and NC del. Novirion-associated RT activity was detected in the CA or RRE deletionclones.

D. Infectivity of the FIV-141 Gene-Deleted Clones In Vitro

CRFK cells were grown in six well plates and transfected as describedpreviously. Forty-eight hours after transfection, 2×10⁶ FeP2 cells wereadded to each well. After co-cultivation of the cells for 72 hours, FeP2cells were separated from CRFK cells and the supernatants from FeP2 cellcultures were harvested and monitored for virus production using the FIVp26 ELISA assay every 3 to 4 days for a total of 4 to 6 weeks.

Twelve deletion clones were found to have no significant level of p26capsid protein expression during the monitoring period. These included:ENV del; TMf del; and NC del (FIG. 5); 3 deletion clones from the Vifregion (Vifn del; Vifc del; and Vif del) (FIG. 6); the MA and CAdeletion clones (FIG. 7); the V3/4, V7/8 and CT deletion clones (FIG.8); and the ORF(2) deletion clone (FIG. 9). The results indicate thatdeletions introduced into these clones totally abolish the infectivityof the virus in the FeP2 cells. Moderate levels of virus replicationwere detected for four deletion clones including DU del; SU del; IN del;and RRE del (FIG. 10).

E. Conclusions

ENV del

The ENV deletion clone, which has a deletion of 701 amino acids in themiddle of the ENV protein (residues 106 to 806), totally lost theability to infect FeP2 cells. Nevertheless, it maintained the ability toassemble and release defective virus particles, to encapsidate the viralgenome, and to reverse transcribe RNA. The primary function of the ENVprotein is to mediate virus entry into target cells during the earlystage of infection. Deletion of the majority of the ENV protein mayblock virus entry and, hence, virus infectivity.

TMf del

The TMf deletion clone, containing a 25 amino acid deletion in thecleavage junction between the SU and TM proteins, is non-infectious inFeP2 cells. The deletion may block the cleavage processing of the ENVprecursor protein, and this may result in a failure of viral particlesto bind to and enter target cells. It has been reported that removal ofthe cleavage site of the ENV glycoprotein of FIV results in theexpression of an uncleaved ENV precursor protein. However, the expressedrecombinant protein maintains its antigenic properties, as evidenced byits interaction with monoclonal antibodies as determined using WesternBlots and radioimmunoprecipitation assays (Rimmelzwaan et al., 1994, J.Gen. Virol. 75:2097-2012). Upon transfection into CRFK cells, thedeletion clone produces defective virus particles at a level comparableto a wild type FIV-141 clone. The defective viral genome and RT enzymeswere encapsidated in the defective virions.

SU del

SU del had a deletion of 503 amino acids from residue 106 to 608 of theSU protein. It was found to maintain levels of virus particle productionapproximately equal to that of the wild type clone. Both thegene-deleted viral genome and RT enzyme were encapsidated. However, incontrast to ENV del, cells transfected by SU del produced virusparticles that are infectious in the FeP2 cells, although to a muchlesser extent than the wild type virus. Thus, it appears that deletionof the SU protein from the FIV-141 genome attenuated the virus. It isbelieved that FIV binding to cellular receptors, which is the first stepin virus infection, is mediated by the SU protein when associated withthe TM protein. The mechanism by which the mutant virus binds to andenters target cells is unknown. An alternative pathway for the mutantvirus to enter host cells may be responsible for the observed lowerinfectivity associated with the deletion clone.

V3/4 del and V7/8 del

One hundred forty-four amino acids from residues 360 to 503 of the SUprotein (covering the V3 and V4 variable regions), and 72 amino acidsfrom residues 98 to 169 of the TM protein (encompassing the V7 and V8regions) were deleted in V3/4 del and V7/8 del respectively. Upontransfection into CRFK cells, each clone produced defective virus atlevels similar to that observed for the wild type clone. As with otherENV-related deletion clones, V3/4 del and V7/8 del encapsidated theirgene-deleted viral genomes and RT enzymes into virions. The infectivityassay indicated that deletion of the V3 and V4 region of SU, anddeletion of the V7 and V8 region in the TM protein, totally abolishedvirus infectivity in the FeP2 cells. The V3 variable region is theimmunodominant domain and has been reported to be involved in multiplefunctions, including virus tropism, viral pathogenesis, and neutralizingepitopes. It is not presently clear at which step viral infection wasblocked in these two deletion clones.

CT del

The TM protein of FIV has a relatively long cytoplasmic tail (46 aminoacids in length). Truncation of this tail in CT del clone resulted in aloss of infectivity of the virus in FeP2 cells. However, truncation hadno effect on virus particle formation and encapsidation of the viralgenome and RT protein. A specific functional interaction between MA andthe TM cytoplasmic tail has been reported for FIV as well as for HIV-1.This interaction has been proposed to be important for the incorporationof the ENV protein into virions. Truncation of the cytoplasmic domain inCT del may eliminate the functional interaction between the MA and TMproteins, thereby blocking the incorporation of ENV.

MA del

MA del contains a 41 amino acid deletion from residues 85 to 125 at theC-terminus of the MA protein. Upon transfection into CRFK cells, theclone produced defective virus at a level comparable to that producedusing the wild type FIV-141 clone. This indicates that deletion at theC-terminus domain has no significant effect on virus particle assemblyand release. The gene-deleted viral genome and RT protein wereencapsidated in the defective virus particles. When these virusparticles were released from transfected CRFK cells, they werenon-infectious with respect to FeP2 cells.

CA del

A deletion of 38 amino acids from residues 9 to 46 at the N-terminus ofCA protein abolished viral particle formation, as evidenced by anegative signal in the p26 ELISA assay, the intra-virion RT PCR assay,and the RT activity assay. However, intracellular RT PCR from thetransfected CRFK cells demonstrated that the deletion did not blockviral RNA expression. Therefore, the failure to detect p26 protein ordefective virus production in the supernatants of transfected cells isdue to the block in viral particle assembly, not in viral proteinexpression.

NC del

The entire NC protein was deleted in the NC del clone. Cells transfectedwith this clone produced defective virus at a significantly reducedlevel compared to the wild type clone, indicating that deletion impairedviral particle assembly or release. It has been reported that the NCprotein of HIV-1 is not required for the assembly of virus-likeparticles. The deletion in the NC clone did not effect the packaging ofthe RT enzyme into defective virions. As expected, no viral genome wasencapsidated in the viral particles.

Vif del, Vifc del and Vifn del

Three deletion clones were constructed in the Vif gene, i.e., Vifn del,Vifc del, and Vif del. Vifn had a deletion of 50 amino acids at theN-terminal portion of the Vif protein. Vifc had a deletion of 146 aminoacids at the C-terminal region of the protein, and Vif del had adeletion of almost the entire Vif protein. All three clones exhibitedsimilar properties. Cells transfected with any of the three clonesproduced virus particles at a comparable rate to the wild type FIV-141clone. Both viral genomes and RT enzyme were encapsidated in virions forall three clones. Virions released from CRFK cells transfected by thethree clones were non-infectious with respect to FeP2 cells, indicatingthat Vif is required for virus replication in T lymphocytes.

ORF(2) del

The entire open reading frame of ORF(2) was deleted in the ORF(2)deletion clone. Cells transfected with the clone assembled and releasedviral particles at a comparable rate to the wild type clone. Althoughboth viral genome and RT enzyme were packaged in the viral particles,the clone failed to replicate in FeP2 cells, suggesting that the geneproduct of ORF(2) is required for virus production in these cells.

RRE del

Eighty-four of the 150 total bases comprising the RRE sequence of FIVwere deleted in RRE del. This deletion severely impaired viralstructural protein expression and the production of viral particles intransfected cells. No p26 production in the supernatants of transfectedCRFK cells was detected. Similarly, no packaged RT activity wasmeasured. These results are in good agreement with the proposal that theRRE sequence is required for the export of unspliced and single-splicedviral RNA from the nucleus to the cytoplasm of cells. However,virion-associated viral genomic RNA was demonstrated to be present by RTPCR and the viral particles were infectious in FeP2 cells, although at amarkedly reduced level compared with the wild type FIV-141 clone. Takenas a whole, these results indicate that deletion of the RRE sequencedramatically decreases the expression of viral structural proteins.However, it appears that the deletion did not totally abolishexpression, and a trace amount of infectious virion particles wasproduced by the transfected cells.

IN del

Almost the entire IN protein was deleted in the IN del clone. Upontransfection into CRFK cells, the clone exhibited a level of viralprotein expression and viral particle production comparable to that ofthe wild type clone. Virion-associated RT PCR and RT activity assaysindicated that both viral genomic RNA and RT enzyme were packaged intoviral particles. Surprisingly, the virions recovered from the cellstransfected with the clone were infectious and could replicate in FeP2cells, although at a reduced level compared with the wild type virus.Integration is an obligate step required for productive infection of anumber of retroviruses, including HIV-1. The data suggest that the INprotein of FIV, in contrast to HIV, may not be an obligate requirementfor viral protein expression and viral replication in FeP2 cells.

DU del

Almost the entire DU gene was deleted in the DU del clone. The productof this gene converts dUTP into dUMP. Deletion of the DU gene in theclone did not affect viral protein expression or viral particleproduction in transfected CRFK cells. Both viral genome and RT enzymewere encapsidated, and the virions produced from transfected cells wereinfectious for FeP2 cells. However, the deletion clone replicated inFeP2 at a slower rate than the wild type FIV-141 virus. This indicatesthat the DU gene is required for maximum replication of the virus. Thedata is consistent with reports that DU deleted FIV maintains itsability to propagate in T lymphocytes.

Example 5 Efficacy of Gene-Deleted FIV-141 Vaccines

Production of Gene-deleted FIV-141 Plasmid DNA for Vaccination

Production of bulk purified plasmid DNA for vaccination was contractedto DNA Technologies, Inc., Gaithersburg, Md. Coded samples of each clonetransformed into Stbl2 E. coli cells were sent to DNA Technologies, andeach clone was grown in approximately 10 liters of LB medium.Supercoiled plasmid DNA was isolated by double CsCl density gradientcentrifugation followed by extensive dialysis. The final purified DNAwas dissolved in phosphate buffered saline (PBS) with 1 mM EDTA at aconcentration of 2-5 μg/μl. Restriction digestion and endotoxin testwere performed for each plasmid DNA preparation.

Vaccination and Challenge

Eleven experimental vaccines were prepared from plasmid DNA describedabove. The appropriate volume of stock DNA from each construct wasdissolved in sterile PBS (GIBCO) to give 300 μg DNA in a 2 mL dose(Table 2). Placebo vaccine was also assembled using the pCR-Script SK(+)vector DNA.

Antibody-profile defined, barrier-reared domestic shorthair cats(approximately 8 weeks of age) were obtained from Liberty Research,Inc.(Waverly, N.Y.). The cats were vaccinated with killed vaccines tofeline herpes virus, feline calicivirus, and feline parvovirus virus.Ten cats were randomly assigned by litter and sex to 13 groups prior tovaccination (Table 2).

TABLE 2 Group Vaccine Vol/Dose Challenge Cat number 1 ENV del 2 ml/300μg Yes 10 2 CA del 2 ml/300 μg Yes 10 3 Vif del 2 ml/300 μg Yes 10 4 INdel 2 ml/300 μg Yes 10 5 ORF(2) del 2 ml/300 μg Yes 10 6 MA del 2 ml/300μg Yes 10 7 Tmf del 2 ml/300 μg Yes 10 8 V3/4 del 2 ml/300 μg Yes 10 9V7/8 del 2 ml/300 μg Yes 10 10 MA del/Tmf del 2 ml/300 μg Yes 10 11 MAdel/V3/4 del 2 ml/300 μg Yes 10 12 Placebo 2 ml/300 μg Yes 10 13 Placebo2 ml/300 μg No 10

Three vaccinations were administered at 4-week intervals when cats were8, 12 and 16 weeks of age. Vaccines were administered into thequadriceps muscle (IM). Each 2 ml dose was divided equally between themuscles on the two hind legs. Four weeks following the last vaccination,all vaccine groups and one placebo group were challenged subcutaneouslyin the nape of the neck with FIV-141 virus at a dose of 354 TCID₅₀ whencats were 20 weeks of age. The second placebo vaccine group received aplacebo challenge of Hank's balanced salt solution. Cats were observedfor 12 weeks post-challenge.

Evaluation of Vaccine Efficacy

Similar to HIV-1 disease progression (Graziosi et al., 1993, Proc. Natl.Acad. Sci 90:6405-6409), FIV RNA load in plasma has been demonstrated tocorrelate with disease stage, and can predict disease progression inaccelerated FIV infection (Diehl et al., 1995, J. Virol. 69:2328-2332;Diehl et al., 1996, J. Virol. 70:2503-2507). In this study, peripheralblood was drawn weekly for monitoring efficacy of the vaccination for 12weeks post-challenge. Plasma viral loads were determined by quantitativecompetitive-reverse transcription-polymerase chain reaction (QcRT-PCR).

1. Quantitation of Viral RNA in Plasma by QcRT-PCR

Viral RNA was isolated from plasma samples using a QIAmp Viral RNAPurification Kit (Qiagen). Each purified RNA sample was distributed intofour tubes, and into each tube was added an internal competitive RNAtemplate with decreasing amounts of RNA (from 1000 fg, 100 fg, 10 fg to1 fg). RNA samples were subjected to RT-PCR using a Titan One TubeRT-PCR System (Boehringer Mannheim). A one-step PCR protocol from themanufacturer was performed with minor modifications to increase thesensitivity of the assay. The RT-PCR reaction was set up in a totalvolume of 38.5 μl containing: 6.5 mM DTT, 0.3 units RNase inhibitor, 0.3mM dATP, 0.3 mM dGTP, 0.3 mM dTTP, 0.3 mM dCTP, 10.4 ng of each FIVspecific oligonucleotide, i.e., QPCR-11 (forward primer1392-TGTAGAGCATGGTATCTTGAAGCATTAGGAAA-1423) (SEQ ID:58) and QPCR-O2(reverse primer 2175-GTTCCTCTCUTTCCGCCTCCTACTCCAATCATATT-2141) (SEQID:59), 1.95 mM MgCl₂, and 1 ul of Titan Enzyme Mix. RT-PCRamplification conditions were 50° C. for 90 min; 94° C. for 3 min;followed by 30 cycles of denaturing at 94° C. for 30 sec, annealing at55° C. for 1 min, and extension at 72° C. for 2 min; followed by 72° C.for 10 min.

Each PCR sample was separated on a 1.0% agarose gel and stained withethidium bromide. Quantitation of viral RNA load was determined bycomparing the intensity of the positive DNA band with that of theinternal competitive standard control DNA band using the Gel-Doc system(Bio-Rad Laboratories).

2. Viral Load in Plasma Post-challenge

Compared with the non-vaccinated (placebo) challenged group, cumulativeviral RNA load in plasma was decreased in most of the vaccinated groupsincluding those vaccinated with FIV-141 ENV del, CA del, V3/4 del, Vifdel, MA del/Tmf del, MA del/V3/4 del, IN del and Tmf del vaccines (FIG.11). The most significant decrease in plasma viral RNA load was achievedin group 1, which was vaccinated with FIV-141 ENV del vaccine. Group 1exhibited a 10-fold decrease in cumulative plasma viral load over aperiod of 12 weeks post-challenge (FIG. 11). Following group 1 inresponse is group 2, which was vaccinated with FIV-141 CA del. Anapproximately 8-fold decease in plasma viral RNA load was observed inthis group. Cats in groups 8, 10, 11 and 3 vaccinated with FIV-141 V3/4del, MA del/Tmf del, MA del/V3/4 del and Vif del, respectively, showed adecrease of approximately 4-fold in plasma viral load. Cats in groups 4and 7 vaccinated with FIV-141 IN del and Tmf del, respectively,exhibited a 2-3 fold decrease in plasma viral load. Viral RNA loads wereslightly decreased in groups 5 and 9 vaccinated with FIV-141 ORF(2) deland V7/8 del vaccine, respectively. Vaccination enhancement of viralinfectivity was observed in group 6, which was vaccinated with FIV-141MA del, where the plasma viral load was increased about 50% over that ofthe non-vaccinated (placebo) challenged group. Thus, decreases in plasmaviral load were demonstrated in several groups vaccinated with a vaccineof the present invention, especially the group vaccinated with FIV-141ENV del vaccine.

Deposit of Biological Materials

The following biological materials were deposited with the American TypeCulture Collection (ATCC) at 12301 Parklawn Drive, Rockville, Md.,20852, USA, on Jul. 1, 1998, and were assigned the following accessionnumbers:

ATCC Accession No. Viral strain FIV-141 VR-2619 Plasmid pFIV-141-B1203001

All patents, patent applications, and publications cited above areincorporated herein by reference in their entirety.

The present invention is not to be limited in scope by the specificembodiments described, which are intended as single illustrations ofindividual aspects of the invention. Functionally equivalentcompositions and methods are within the scope of the invention.

59 1 9464 DNA Feline immunodeficiency virus 1 tgggaagatt attgggatcctgaagaaata gaaaaaatgc taatggactg aggacgtaca 60 taaacaagtg acagatggaaacagctgaat atgactcaat gctagcagct gcttaaccgc 120 aaaaccacat cctatgtaaagcttgccgat gacgtgtatc ttgctccatt ataagagtat 180 ataaccagtg ttttgtaaaagcttcgagga gtctctctgt tgagggcttt cgagttctcc 240 cttgaggctc ccacagatacaataaaaaac tgagctttga gattgaaccc tgtcttgtat 300 ctgtgtaatt tctcttacctgcgaatccct ggagtccggg ccagggacct cgcagttggc 360 gcccgaacag ggacttgaaaaggagtgatt agggaagtga agctagagca atagaaagct 420 gtcaagcaga actcctgcaggccttgtatg gggagcagtt gcagacgctg ctggcagtga 480 gtatctctag tggagcggacctgagctctg gattaagtca ctgctcacag gcctagataa 540 agattatctg gtgactcttcgcggatcgtc aaaccagggg attcgtcggg ggacagccaa 600 caaggtagga gagattctacagcaacatgg ggaatggaca ggggcgagac tggaaaatgg 660 ccattaagag atgtagtaatgttgctgtag gggtagggag caggagtaaa aaatttggag 720 aaggaaattt tagatgggccataaggatgg ctaatgtaac tacaggacga gaacctggtg 780 atataccaga gactttagaacagctaagat caatcatttg tgacttacaa gacagaagag 840 aacaatatgg atctagtaaagaaattgaca tggcaattac cactttaaaa gtttttgcag 900 tggcaggaat tctaaatatgactgtaacta ctgccacagc agctgaaaat atgtatgctc 960 agatgggatt agacaccagaccatctataa aagaaagtgg gggaaaagaa gaaggacctc 1020 cacaggctta tcctattcaaacagtaaatg gagcaccaca gtatgtagcc cttgatccaa 1080 aaatggtgtc tatttttatggagaaggcaa gagaggggct aggaggtgaa gaagtccaac 1140 tgtggtttac agccttttcagctaatttaa catcaactga tatggctaca ttaattatgt 1200 ccgcacctgg ctgtgcagcagataaagaaa tcctagatga aacactgaaa cagatgacag 1260 ctgagtatga tcgtacccatcctcctgatg ggcctagacc gctgccctat ttcactgccg 1320 cagagatcat ggggataggattgactcaag aacaacaagc agaacccagg tttgccccag 1380 ccagaatgca gtgtagagcatggtatcttg aagcattagg aaagctagcg gccataaaag 1440 ccaaatctcc ccgagcagtacaattgaagc agggagctaa agaggactat tcctcattca 1500 tagatagact atttgctcaaatagatcaag agcagaacac agctgaggta aagctgtatt 1560 taaaacaatc tttgagcatagcaaatgcta atccagattg taagagagcg atgagtcatc 1620 ttaaaccaga aagtactttagaagagaaac tgagagcctg ccaggaaata ggatcgccag 1680 gatacaaaat gcaactattggcagaggctc ttactagggt gcaaacagtt caagcaaaag 1740 gaccaaggcc agtatgtttcaattgtaaaa aaccaggaca cctggccaga caatgtagac 1800 aagcaaagag atgtaataaatgtggaaaac ctggtcactt agctgctaac tgttggcaag 1860 gaggtaaaaa gtccccgggaaacggggcga tggggcgagc tgcagcccca gtaaatcaag 1920 tgcagcaagt gataccatctgcacccccgg tagaggagaa attgttagat atgtaaacta 1980 taataaagtg ggtaccaccacaactttaga aaaaagacct gaaatacaaa tattcgtaaa 2040 tgggtatcct ataaaatttttattagatac aggagcagat ataacaattt taaacagaaa 2100 agactttcag atagggaattctatagaaaa tgggaaacag aatatgattg gagtaggagg 2160 cggaaagaga ggaacaaattatatcaatgt gcatttagaa attagagatg aaaattataa 2220 gacacagtgt atatttggaaatgtgtgtgt cttggaggat aattcattaa tacaaccatt 2280 attgggaaga gataacatgattaagttcaa cataaggttg gtaatggctc aaatttcaga 2340 gaaaattcca atagtaaaagtaagaatgaa agaccctact caagggcctc aggtaaaaca 2400 atggccatta tcaaatgagaaaattgaagc tctaactgac atagtaaaca ggttagaaca 2460 agagggaaag gtaaaaagagctgatccaaa taatccttgg aacactcccg tatttgcaat 2520 caagaaaaag aatggtaaatggagaatgct catagatttt agggtcctaa ataaattaac 2580 agacaaaggg gcagaagttcagttaggact ccctcatcct gctggattac aattgaaaaa 2640 acaagtaact gtattggacataggggacgc atattttact attcctctag atccagatta 2700 tgctccttat actgcatttacactacctag aaaaaacaat gcaggaccag ggaggagata 2760 catatggtgt agtttaccacaagggtgggt cttgagtcca ttgatatatc agagtacctt 2820 agacaatata ctccaaccttttattaaaca gaatcctgag ttagatattt atcaatatat 2880 ggatgatatc tatataggatcaaatttaag taaaaaggaa cataaactaa aagtagaaga 2940 attaagaaaa ttgttattatggtggggatt tgaaaccccg gaagataaat tacaagaaga 3000 gcccccctat aagtggatgggctatgaatt acatccatta acgtggtcaa tacagcaaaa 3060 gcaattagaa attccagagagacccacatt aaatgaatta cagaagttag caggtaagat 3120 taactgggct agtcaaaccattccagactt gagcataaaa gaactaacta atatgatgag 3180 aggagatcaa aagttagactcaataagaga atggacgaca gaggccaaga atgaagtgga 3240 gaaagctaag agagcaattgagacacaggc acagctagga tattatgatc ctaatcgaga 3300 attatatgct aaattaagtcttgtgggacc acatcaacta agctatcagg tgtatcataa 3360 aaacccagaa cagatattatggtatgggaa aatgaatagg cagaagaaaa aagcagaaaa 3420 tacttgtgat atagctctaagggcatgtta caaaataaga gaagaatcca ttataagaat 3480 aggaaaagaa ccagtatatgaaatacctac atccagagaa gcttgggaat caaatctaat 3540 tagatctcca tatcttaaggcctcaccacc tgaggtggaa tttatacatg ctgccttaaa 3600 tataaaaaga gctctaagcatgatacaaga tgcccctata ttgggagcag aaacatggta 3660 catagatggg ggaagaaaacaaggaaaagc agcaagagca gcttattgga cagatacggg 3720 cagatggcag gtaatggaaatagaaggaag taatcaaaaa gcagaagtac aagctttatt 3780 attggcccta caggcaggaccagaggaaat gaatattata acagattcac aatatattgt 3840 gaatattatt aatcaacaaccagatttgat ggaaggaatt tggcaagaag tcttagaaga 3900 aatggaaaag aaagtagcaatctttataga ttgggtacct ggacataaag gtattccagg 3960 aaataaagag gtagatgaactttgtcaaac gatgatggtt atagaaggtg aaggaatatt 4020 agataaaaga tcagaagatgcaggatatga tttattagct gcacaagaaa tacatctctt 4080 gcctggggag gtaagagtagtaccaacaag aacaaagata atgttaccta aaggatattg 4140 gggattaata atgggaaaaagttcaatggg aagcaaagga ttagatgtat taggaggagt 4200 tatagatgaa ggatatagaggagaattagg ggtgataatg attaacctat ctaaaaaatc 4260 aataacatta tcagaaaaacaaaaagtagc acaattaata atattacctt gtaaacatga 4320 aagcttacaa caaggagaaataataatgga ttcagaaaga ggaagaaagg gatttgggtc 4380 aactggagtc ttttcttcatgggtggacag aattgaggaa gcagaattaa atcatgaaaa 4440 atttcactca gacccacaatacttaagaac agaatttaat ctacccagaa tagtagcaga 4500 ggaaataaaa agaaaatgtcccttatgtag aatcagaggg gaacaagtag ggggacaatt 4560 aaagattgga cctggcatatggcaaatgga ctgtacacac tttaatggaa aaataattat 4620 tgtcgcagtg catgtggaatcaggcttatt atgggcacag gtaattccac aggagactgc 4680 agattgtaca gttaaagctctcatgcaact tatcagtgct cataatgtta cagaactaca 4740 aacagataat ggaccaaattttaaaaatca gaaaatggaa ggactactaa attatatggg 4800 cataaaacac aaattaggtataccaggtaa cccacaatca caagcattag tagaaaatgc 4860 taaccacaca ttaaaatcttggattcaaaa atttctctca gaaacttctt ctttggacaa 4920 cgcattggcc ctagccttatactgcctcaa ttttaaacaa aggggtagac tagggagaat 4980 ggctccttat gaattatacatacaacagga atcattaaga atacaagact atttttcaca 5040 aattccacaa aaattaatgatgcaatgggt gtattataaa gatcagaaag ataaaaagtg 5100 gaagggacca atgagagtagaatattgggg acaaggatca gtattattaa agaatgaaga 5160 gaagggatat tttcttgtacctaggagaca cataagaaga gtcccagaac cctgcactct 5220 tcctgaaggg gatgagtgacgaagattggc aggtaagtag aagactcttt gcagttctcc 5280 aaggaggagt aaatagtgccatgttataca tatcgaattt acctgaaaca gaacaggcac 5340 aatataaaaa ggactttaagaaaaggctct tagaaaagga gactggattc atctatagat 5400 taagaaaagc tgaaggaataaggtggagct ttcatacgcg tgattattat ataggatatg 5460 taagagagat ggtggctgggtctagcctac aaaatagttt aagattgtat gtttatataa 5520 gcaatccatt gtggcatcagtcataccgtc ctggcctgac aaattttaat acagagtggc 5580 cttttgtaaa tatgtggataaagacaggat ttatgtggga tgatattgaa agccaaaata 5640 tttgcaaagg aggagagatctcacatggat ggggacctgg aatggtggga attgtgataa 5700 aagcatttag ctgtggagaaaggaagatac aaattactcc tgtcatgatt ataagaggtg 5760 agatagaccc acagaaatggtgtggagatt gttggaatct gatgtgtctt aaatattcac 5820 ttccaaatac attgcagaggcttgctatgc tggcgtgtgg caaagaggct aaagaatgga 5880 gaggctgttg taatcagcgttttgtttctc ctttcagaac accctgtgat ctagaggtcg 5940 tccagaacaa gcctaaaaggaatttattgt ggacgggaga attatgaatg gaagaaataa 6000 tcccactgtt taataaggttacagaaaagt tagatagaga agcagctatt agattgttta 6060 ttttagctta tcaggtagacagatgcagat ttattagaat tttacaatta ttactttgga 6120 gagatagatt taagtcaatcaattctaaat attgtttatg ctggctgtgc tgcaagtctg 6180 cttattggcg cttgcaatctacattatcca taaatactgc ctagaaatat ttcttttaat 6240 atttcatctg cagatataaacatggcagag ggaggattta ctcaaaatca acaatggata 6300 gggccagaag aagctgaagaattgttagat tttgatatag ctgtacaaat gaatgaagaa 6360 ggtccattaa acccaggagtaaacccattt agggtaccag gaattacctc tcaagaaaag 6420 gatgattatt gtcagattttacaaccaaaa ctacaagaat taaagaatga aatcaaagag 6480 gtaaaacttg acgaaaacaatgcaggtaag tttagaaagg caagatattt aagatattct 6540 gatgagagtg tactaactatagtctattta ctaacaggat atttgagata tttaataagc 6600 catagaaact taggatctttaagacatgat atagatatag aagcaccaca acaagagcac 6660 tataatgata aagaaaagggtactacttta aatataaagt atgggagaag atgttgtatt 6720 agcacattac ttctatatttaatcctcttc tcagggatag gaatttggct tggaaccaaa 6780 gcacaagtag tgtggagactccctccttta gtagtgccag tagatgagac agaaataata 6840 ttttgggatt gttgggcgccagaggaacca gcctgtcaag attttctggg aacaatgata 6900 catttaaaag caaatgttaatataagtata caagaaggac ctacattggg aaattgggca 6960 agggaaattt ggtctacattatttaaaaaa gctacaaggc aatgcagaag gggaaggata 7020 tggaagaaat ggaatgagactataacagga cctaaaggat gtgcaaataa tacctgttat 7080 aatatttcag tagtggtacctgattatcaa tgttatgtag acagagtaga tacatggctg 7140 caaggaaaag ttaatatctcactatgtttg acaggaggaa agatgctata taataaaaat 7200 acaaaacaat taagttactgtacagatcca ttacaaatac cattaattaa ttacacattt 7260 ggacctaacc aaacttgtatgtggaacaca tctttaatca aagaccctga gataccgaaa 7320 tgtggatggt ggaaccaggcagcctattat aataattgta aatgggaaga agctaatgtg 7380 acatttcaat gtcaaagatcacaaagtcta ccaggatcat gggttaggag aatctcttca 7440 tggagacaaa gaaacagatgggagtggagg ccagactttg aaagtgagaa agtaaaaata 7500 tcattacaat gtaatagtacaaaaaattta acttttgcaa tgagaagttc aagtgattat 7560 tatgatgtac aaggagcatggatagaattt ggatgttata gaaataaatc aagaacccat 7620 acgggagcaa gatttagaataagatgtaaa tggaatgaag gaaagaatct atctctcatt 7680 gatacatgtg ggactacttcaaatgtgaca ggagccaacc ctgtagattg tactatgaaa 7740 acaagcacta tgtacaattgttccttacaa gatagtttca ctatgaaaat agaggacctt 7800 attgtacaat ttaatatgacaaaagcagtg gaaatgtata atattgctgg gaattggtct 7860 tgtacatctg atttaccaacagggtgggga tatatgaaat gtaattgtac aaatgccact 7920 gatggggaga ataaaatgaaatgccctagg aatcagggta ttttaagaaa ctggtacaat 7980 ccagttgcag gactaagacaagctcttatg aagtatcaag tagtaaaaca accagaatat 8040 ttggtggtac cggaagaagttatgaggtat aaaggtaaac aaaaaagggc cgctattcat 8100 attatgttag cccttgctacggtgttatct atagctggag caggaaccgg tgccactgct 8160 attgggatgg tgacacactatcagcaagtt ttggctaccc atcagcaggc attggacaaa 8220 ataactgagg cactgaaaataaacaactta aggttaatca ctttagaaca tcaagtatta 8280 gtgatagggt taaaagtagaggctatagaa aaattcctat atacagcttt tgctatgcaa 8340 gaattaggat gtaatcagaatcaattcttt tgtaagattc ccctcaatct gtggacaatg 8400 tataacatga ctataaatcatacactatgg aatcatggaa atataacttt gggagaatgg 8460 tataatcaaa caaaaagtttacaagaaaaa ttttatgaga taattatgga tatagaacaa 8520 aataatgtac aagggaaaaatggaatacaa caattacaaa aatgggaaaa ttgggtggga 8580 tggataggca aaatccctcaatatttaaaa ggacttcttg gtagtgtgtt gggaatagga 8640 ctaggaatct tactactacttatatgcttg cctacattag tagattgtat aagaaactgt 8700 actaataaaa tattgggatatacagttatt gcaatgcctg aaatagatga tgaggaagta 8760 cacccatcag tggaattgaggagaaatggc aggcaatgtg gcatatctga aaaagaggag 8820 gaatgatgga gcatttcagacctgtagaat acaggagtaa tgctgagctg agttcttccc 8880 tttgaggagg atgtgtcatatgaatccatt tcaaatcaaa aataacagta aaatctatat 8940 tgtaaggcaa acgaaaaagacaacgcagaa gaagaaagaa gaaggccttc aaaaaattga 9000 tgctggattt agaggctcgatttaaagcgt tgtttgaaac accttcagct acagaatata 9060 ctgcagacga gacagaagaagagactcttg aaaaagaaaa aagggtggac tgggaagatt 9120 attgggatcc tgaagaaatagaaaaaatgc taatggactg aggacgtaca taaacaagtg 9180 acagatggaa acagctgaatatgactcaat gctagcagct gcttaaccgc aaaaccacat 9240 cctatgtaaa gcttgccgatgacgtgtatc ttgctccatt ataagagtat ataaccagtg 9300 ttttgtaaaa gcttcgaggagtctctctgt tgagggcttt cgagttctcc cttgaggctc 9360 ccacagatac aataaaaaactgagctttga gattgaaccc tgtcttgtat ctgtgtaatt 9420 tctcttacct gcgaatccctggagtccggg ccagggacct cgca 9464 2 30 DNA Feline immunodeficiency virus 2ccgcaaaacc acatcctatg taaagcttgc 30 3 30 DNA Feline immunodeficiencyvirus 3 cgcccctgtc cattccccat gttgctgtag 30 4 30 DNA Felineimmunodeficiency virus 4 ttactgtttg aataggatat gcctgtggag 30 5 30 DNAFeline immunodeficiency virus 5 gcaatgtggc atgtctgaaa aagaggagga 30 6 34DNA Feline immunodeficiency virus 6 tcttcccttt gaggaagata tgtcatatgaatcc 34 7 26 DNA Feline immunodeficiency virus 7 tctgtgggag cctcaagggagaactc 26 8 30 DNA Feline immunodeficiency virus 8 acaaacagat aatggaccaaattttaaaaa 30 9 28 DNA Feline immunodeficiency virus 9 tttcaatatcatcccacata aatcctgt 28 10 30 DNA Feline immunodeficiency virus 10ttaaaggatg aagagaaggg atattttctt 30 11 30 DNA Feline immunodeficiencyvirus 11 tgggaagatt attgggatcc tgaagaaata 30 12 40 DNA Felineimmunodeficiency virus 12 catatcctat ataataatca cgcgtatgaa agctccacct 4013 24 DNA Feline immunodeficiency virus 13 tgcgaggtcc ctggcccgga ctcc 2414 40 DNA Feline immunodeficiency virus 14 aggtggagct ttcatacgcgtgattattat ataggatatg 40 15 29 DNA Feline immunodeficiency virus 15ctccagggat tcgcaggtaa gagaaatta 29 16 49 DNA Feline immunodeficiencyvirus 16 ttacaagaat tcaactgcag tgggaagatt attgggatcc tgaagaaat 49 17 42DNA Feline immunodeficiency virus 17 ttcaaggagc tcttttgtcg acaactgcgaggtccctggc cc 42 18 53 DNA Feline immunodeficiency virus 18 gattcgtcgggggacagcca acaaggtagg agagattcta cagcaacatg ggg 53 19 42 DNA Felineimmunodeficiency virus 19 tcaatatatg gatgatatct atataggatc aaatttaagt aa42 20 58 DNA Feline immunodeficiency virus 20 gtgatatagc tctaagggcatgttacaaaa taagagaaga atccattata agaatagg 58 21 46 DNA Felineimmunodeficiency virus 21 cgggcagatg gcaggtaatg gaaatagaag gaagtaatcaaaaagc 46 22 44 DNA Feline immunodeficiency virus 22 agaaagggatttgggtcaac tggagtcttt tcttcatggg tgga 44 23 51 DNA Felineimmunodeficiency virus 23 gggggacaat taaagattgg acctggcata tggcaaatggactgtacaca c 51 24 49 DNA Feline immunodeficiency virus 24 ggctccttatgaattataca tacaacagga atcattaaga atacaagac 49 25 36 DNA Felineimmunodeficiency virus 25 caaaatagtt taagattgta tgtttatata agcaat 36 2640 DNA Feline immunodeficiency virus 26 cagaaaagtt agatagagaa gcagctattagattgtttat 40 27 40 DNA Feline immunodeficiency virus 27 taaaagcaaatgttaatata agtatacaag aaggacctac 40 28 40 DNA Feline immunodeficiencyvirus 28 aaaagctaca aggcaatgca gaaggggaag gatatggaag 40 29 44 DNA Felineimmunodeficiency virus 29 agaggacctt attgtacaat ttaatatgac aaaagcagtggaaa 44 30 40 DNA Feline immunodeficiency virus 30 ccctcaatct gtggacaatgtataacatga ctataaatca 40 31 40 DNA Feline immunodeficiency virus 31gacaacgcag aagaagaaag aagaaggcct tcaaaaaatt 40 32 50 DNA Felineimmunodeficiency virus 32 agtaaagaaa ttgacatggc gattactagt ttaaaagtttttgcagtggc 50 33 47 DNA Feline immunodeficiency virus 33 ccatctataaaagaaagtgg gactagtgaa gaaggacctc cacaggc 47 34 51 DNA Felineimmunodeficiency virus 34 attcaaacag taaatggagc aactagttat gtagcccttgatccaaaaat g 51 35 51 DNA Feline immunodeficiency virus 35 acagccttttcagctaattt aactagtact gatatggcta cattaattat g 51 36 50 DNA Felineimmunodeficiency virus 36 actatagtct atttactaac tggttacctg agatatttaataagccatag 50 37 54 DNA Feline immunodeficiency virus 37 tacttatatgcttgcctaca ttgggttacc gtataagaaa ctgtactaat aaaa 54 38 54 DNA Felineimmunodeficiency virus 38 gaggtataaa ggtaaacaaa aaactagtgc cattcatattatgttagccc ttgc 54 39 58 DNA Feline immunodeficiency virus 39 actaactatagtctatttac taacaactag tttgagatat ttaataagcc atagaaac 58 40 62 DNA Felineimmunodeficiency virus 40 ataccgaaat gtggatggtg gaatcaggca tgctattataataattgtaa atgggaagaa 60 gc 62 41 58 DNA Feline immunodeficiency virus41 gcactatgta caattgttcc ttacaggcat gcttcactat gaaaatagag gaccttat 58 4256 DNA Feline immunodeficiency virus 42 gaatcaattc ttttgtaaga tcgcatgcaatctgtggaca atgtataaca tgacta 56 43 61 DNA Feline immunodeficiency virus43 gggaaaattg ggtgggatgg ataggtaaga tcgcatgcta tttaaaagga cttcttggta 60g 61 44 40 DNA Feline immunodeficiency virus 44 ggaagaagtt atgaggtataccggtaaaca aaaaagggcc 40 45 62 DNA Feline immunodeficiency virus 45ctacttatat gcttgcctac attggtcgac tgatagtgaa actgtactaa taaaatattg 60 gg62 46 56 DNA Feline immunodeficiency virus 46 gatggttata gaaggtgaaggaattactag taaaagatca gaagatgcag gatatg 56 47 59 DNA Felineimmunodeficiency virus 47 gaaataataa tggattcaga aagaggaact agtggatttgggtcaactgg agtcttttc 59 48 57 DNA Feline immunodeficiency virus 48cttcatgggt ggacagaatt gaaactagtg tattaaatca tgaaaaattt cactcag 57 49 59DNA Feline immunodeficiency virus 49 gcaatgggtg tattataaag atcagactagtaaaaagtgg aagggaccaa tgagagtag 59 50 57 DNA Feline immunodeficiencyvirus 50 agaagactct ttgcagttct ccaatgaacg cgttagagtg ccatgttata catatcg57 51 44 DNA Feline immunodeficiency virus 51 cgtgtggcaa agaggctaaaacgcgtagag gctgttgtaa tcag 44 52 56 DNA Feline immunodeficiency virus 52gtggacggga gaattatgaa cgcgtgaact aatcccactg tttaataagg ttacag 56 53 55DNA Feline immunodeficiency virus 53 ctacattatc cataaatact gcctagacgcgtttctttta atatttcatc tgcag 55 54 54 DNA Feline immunodeficiency virus54 ggcatatctg aaaaagagga ggaatgaact agtatatcag acctgtagaa taca 54 55 59DNA Feline immunodeficiency virus 55 gaggaggatg tgtcatatga atcaaatactagtcaaaaat aacagtaaaa tctatattg 59 56 22 DNA Feline immunodeficiencyvirus 56 tattatggtg gggatttgaa ac 22 57 22 DNA Feline immunodeficiencyvirus 57 taattagatt tgattcccag gc 22 58 32 DNA Feline immunodeficiencyvirus 58 tgtagagcat ggtatcttga agcattagga aa 32 59 35 DNA Felineimmunodeficiency virus 59 gttcctctct ttccgcctcc tactccaatc atatt 35

What is claimed is:
 1. An attenuated FIV-141 virus for use as a vaccinewherein said virus replicates upon entry into a host cell but whichexhibits significantly reduced infectivity to feline T lymphocytes whencompared to the wild type FIV-141 virus, wherein said attenuated virusis produced by deletion mutation in the ENV gene.
 2. A host cellinfected with the attenuated virus of claim
 1. 3. An attenuated wholevirus vaccine, comprising the virus of claim 1 and a pharmaceuticallyacceptable carrier.
 4. A purified FIV-141 nucleic acid molecule for useas a vaccine, said molecule having a nucleotide sequence comprising SEQID NO: 1, but wherein the ENV gene of said nucleic acid molecule ismutated by deletion such that when the mutated nucleic acid molecule hasbeen introduced into a host cell, the host cell produces an attenuatedvirus that exhibits greater efficacy against wild type FIV-141 virusinfection.
 5. A host cell transfected with the nucleic acid molecule ofclaim
 4. 6. A vaccine comprising the nucleic acid molecule of claim 4 ata concentration sufficient to induce immunity when administered to acat, and a pharmaceutically acceptable carrier.
 7. A method of inducingthe production of antibodies FIV-141 in cats, comprising administeringto said cat: (a) a substantially purified inactivated FIV-141 virus; (b)a host cell infected with said substantially purified inactivatedFIV-141 virus; (c) an attenuated FIV-141 virus; (d) a host cell infectedwith said attenuated FIC-141 virus; (e) a nucleic acid molecule encodingan attenuated FIC-141 virus; or (f) a host cell transfected with anucleic acid molecule encoding an attenuated FIV-141 virus.
 8. Themethod of claim 7, further comprising purifying the antibody from thecat.